Methods and compositions for modulating RAD51 and homologous recombination

ABSTRACT

The present invention concerns methods and compositions involving inhibitors and enhancers of RAD51, a protein involved in homologous recombination. In some embodiments, the present invention concerns methods for stimulating homologous recombination, which has a number of significant research and clinical applications. In certain other embodiments, there are methods for protecting cells using a compound that enhances RAD51 activity. Such enhancers may also be employed to prevent or reduce damage to cells that may be caused by DNA damaging agents. In other embodiments, there are methods for sensitizing cells to the effects of DNA damaging agents, which can have particular applications for cancer patients. In some embodiments of the invention, the RAD51 enhancer or inhibitor is a small molecule that directly affects RAD51 activity, such as its ability to promote filament formation.

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2008/071364 filed Jul. 28, 2008which claims priority to U.S. Provisional Application No. 60/952,565filed on Jul. 28, 2007, U.S. Provisional Patent Application 60/972,593filed on Sep. 14, 2007, U.S. Provisional Patent Application 61/024,497filed on Jan. 29, 2008, and U.S. Provisional Application No. 61/024,513filed on Jan. 29, 2008, each of which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biochemistry,cell biology, and oncology. More specifically, it concerns methods formodulating RAD51 protein activity in a cell.

2. Description of Related Art

Homologous recombination (HR) has multiple roles in DNA repair includingthe repair of DNA double-strand breaks (DSBs) and recovery from thereplication blocking lesions formed by DNA cross-linking agents. HRrepairs DSBs by locating a homologous stretch of DNA and replicating themissing genetic information from the homologous template. In contrast torepair by non-homologous end joining (NHEJ), DSB repair by HR generallyoccurs without mutations (Thompson and Schild, 2001). Numerous studieshave also shown HR to be critically important in the maintenance ofgenomic stability (Thompson and Schild, 2001; Godthelp et al., 2002;Tebbs et al., 1995; Takata et al., 2001; Liu et al., 2002; Cui et al.,1999; Thompson and Schild, 1999). The proposed mechanism for thispathway begins with 5′ to 3′ nuclease activity at the DSB, resulting ina 3′ single stranded tail (FIG. 1). The tail is coated by ReplicationProtein A (RPA), which is subsequently replaced by a helical filament ofRAD51 protein. This displacement of RPA by RAD51 appears to be mediatedby a number of protein complexes, which include RAD52 and a family ofRAD51 paralog proteins (Thompson and Schild, 2001; Gasior et al., 2001).These RAD51 filaments can be microscopically visualized with fluorescentantibodies, and they appear as sub-nuclear foci (Bishop et al., 1998).The RAD51 coated 3′ tail then invades a double stranded stretch ofhomologous template DNA. The genetic code is essentially copied fromthis template by polymerase activity and branch migration, in astructure termed the Holliday junction.

In addition to RAD51, repair via HR requires five RAD51 paralogproteins. The paralogs form two complexes in solution, a XRCC3/RAD51Cheterodimer and a RAD51B/RAD51C/RAD51/XRCC2 heterotetramer (Liu et al.,2002; Masson et al., 2001a; Masson et al., 2001b; Wiese et al., 2002).Mutation of any one of the five paralog genes prevents subnuclearassembly of recombinase at damaged sites and reduces the cell's abilityto perform HR repair (Godthelp et al., 2002; Tebbs et al., 1995; Takataet al., 2001; Bishop et al., 1998; Fuller and Painter, 1988; Caldecottand Jeggo, 1991; Liu et al., 1998). These paralogs are thought to serveas assembly ‘mediators’ for RAD51. RPA can inhibit assembly of RAD51recombinase at sites of damage, and mediator proteins are thought tohelp overcome this inhibition. Studies have demonstrated a sensitizationto certain DNA damaging therapies associated with cellular defects inproteins that promote HR DNA repair. This sensitization is particularlydramatic for DNA cross-linking chemotherapeutic drugs (30-100 times) andionizing radiation (3-5 times) (Godthelp et al., 2002; Tebbs et al.,1995; Takata et al., 2001; Liu et al., 1998).

Several groups have recently demonstrated that HR can be partiallyinhibited in order to sensitize cells to DNA damaging therapies.Inhibition of XRCC3 (a RAD51 paralog protein), has been demonstratedusing a synthetic peptide corresponding to another paralog protein. Thispeptide sensitized Chinese Hamster Ovary (CHO) cells to cisplatin andinhibited the formation of sub-nuclear RAD51 foci in response to DNAdamage (Connell et al., 2004). Other researchers have inhibited theexpression of the RAD51 protein itself (Russell et al., 2003; Hansen etal., 2003; Ohnishi et al., 1998; Ito et al., 2005; Collis et al., 2001)or blocked its function by over-expressing a dominant negative BRCpeptide fragment derived from BRCA2 (Chen et al., 1999).

In view of the connection between increased sensitivity to certain DNAdamaging therapies and cellular defects in HR DNA repair-relatedproteins, methods and compounds that provide for selective manipulationof this balance are desirable.

SUMMARY OF THE INVENTION

The present invention is based on the identification andcharacterization of compounds that alter the ability of the RAD51protein to bind fluorescently labeled oligonucleotides. Therefore, thepresent invention concerns methods and compositions involving compoundsthat directly modulate the activity of the RAD51 protein. In certainembodiments, methods and compositions concern compounds that modulateRAD51 activity by increasing, enhancing, or stimulating RAD51 activity.In other embodiments, methods and compositions concern compounds thatmodulate RAD51 activity by decreasing, inhibiting, or reducing RAD51activity.

In certain embodiments, there are methods for modulating a RAD51 proteinin a cell comprising providing to the cell an effective amount of aRAD51 protein modulator, wherein the modulator is a small molecule thatdirectly modulates the activity of RAD51 protein. This means that smallmolecules of the invention alter the activity of RAD51 protein (or aRAD51 protein analog or homolog) directly (i.e., RAD51 activityincreases when the RAD51 protein is contacted or incubated with thesmall molecule), and not indirectly, such as by altering the expressionlevel of RAD51.

In some embodiments of the invention, methods and compositions concern acompound that is a RAD51 enhancer, meaning that the compound directlyincreases, enhances, and/or stimulates RAD51 protein activity when theRAD51 protein is exposed to the compound. The terms “enhancer” and“stimultor” are used interchangeably herein. In particular embodiments,the RAD51 enhancer increases RAD51 filament formation when the enhanceris incubated with RAD51 protein and nucleic acid molecules underconditions to promote filament formation. In certain other embodiments,the RAD51 enhancer increases or stimulates homologous recombination viaRAD51 in a cell. In even further embodiments, the RAD51 enhancerprevents or reduces damage to DNA in a cell. In additional embodiments,the RAD51 enhancer prevents or reduces cell death insofar as RAD51activity can contribute to that effect. The present invention alsoconcerns embodiments in which a RAD51 enhancer prevents or reducesmutations in a cell.

Therefore, the present invention is contemplated to cover a number ofmethods involving a RAD51 enhancer, which may increase, enhance orstimulate a RAD51 activity by or by at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more (and any rangederivable therein) compared to RAD51 activity in the absence of theRAD51 enhancer. In certain embodiments there are methods for increasingRAD51 activity in a cell involving providing to the cell an effectiveamount of a RAD51 enhancer.

Some embodiments of the invention involve methods for preventing orreducing DNA damage in cells comprising providing to the cells aneffective amount of a RAD51 enhancer, wherein the RAD51 enhancerdirectly increases RAD51 activity. In particular embodiments it iscontemplated that the damage may be from a treatment such aschemotherapy or radiation or from environmental exposure to a DNAdamaging agent. In some situations it is contemplated that the DNAdamaging agent may be from an industrial accident or from a weapon, suchas a bomb (“dirty bomb”).

In certain embodiments, there are methods for stimulating homologousrecombination in cells comprising providing to the cell an effectiveamount of a RAD51 enhancer that directly increases the activity of RAD51or a RAD51 homolog in the cells.

In additional embodiments there are methods for reducing the incidenceof mutations in a cell comprising providing to the cell an effectiveamount of a RAD51 enhancer such that the number of mutations isdecreased. It is contemplated that some mutations may lead to geneticdefects or birth defects and therefore, the present invention may beapplied in the context of sex cells, fetal cells, and embryo cells. Insome cases, the RAD51 enhancer could be provided to cells systemically.

In other embodiments, there are methods for incorporating exogenous DNAinto cells' DNA comprising: a) transfecting the cells with a compositioncomprising the exogenous DNA; and, b) contacting the cells with aneffective amount of a RAD51 enhancer.

The present invention also concerns methods for increasing theefficiency of gene therapy in a patient comprising administering genetherapy to the patient and an effective amount of a RAD51 enhancer thatdirectly increases RAD51 activity.

In some embodiments it is contemplated that methods of the inventioninvolving a RAD51 enhancer may be used for gene targeting (altering ordeleting a gene through recombination using an exogenous sequence) orfor gene conversion (nonreciprocal transfer of genetic informationthrough recombination). Such embodiments may be implemented byintroducing into a cell a nucleic acid that is to be used for the genetargeting or for the gene conversion and by exposing the cell to aneffective amount of a composition comprising a RAD51 enhancer. In someembodiments involving gene targeting, the nucleic acid containssequences adjacent to the sequence to be deleted (knocked out). In otherembodiments the nucleic acid contains a sequence that an endogenoussequence will be converted to. In specific embodiments, the exogenousnucleic acid contains one or more mutations relative to an endogenoussequence. In other embodiments, the exogenous nucleic acid has awild-type sequence relative to an endogenous sequence, and such methodscan be used to correct genetic defects.

The present invention also covers methods in which homologousrecombination is stimulated to alter genetic map distances, such as forimproving the efficiency or resolution of gene mapping. In suchsituations a RAD51 enhancer can be employed. In other embodiments, itmay be advantageous to inhibit homologous recombination so as to altergenetic map distances. A RAD51 inhibitor could be used for these typesof situations.

Other embodiments involving the use of an effective amount of RAD51enhancer to promote recombination within a cell that contains aheterologous nucleic acid sequence already incorporated in a cell's DNA.In some cases, the heterologous nucleic acid sequence has beenincorporated into a random site in the DNA and this may be employed tore-position the heterologous sequence or better target it to aparticular site. In particular embodiments, such methods may be employedin any variety of cells, including human cells or Drosophilamelanogaster cells.

A RAD51 enhancer is a compound that acts in conjunction with RAD51protein to increase, enhance, or stimulate the activity of the RAD51protein. In certain embodiments the RAD51 enhancer is a small molecularweight compound. In specific embodiments, the RAD51 stimulator is acompound of formula (VII):

or a salt thereof, wherein R₄₅ is H, —C(O)(CH₂)_(q)NH-aryl,—NHC(O)O-alkyl, or

wherein R₄₉ is halogen or alkoxy; Z₂₀ is O or S; and q is 0-6; R₄₆ is Hor halogen; R₄₇ is H, alkyl, hydroxy, or halogen; R₄₈ is H, hydroxy, or—SO₂NH(CH₂)_(r)-aryl, wherein r is 0-6; and R₅₀ is H or hydroxy. Incertain embodiments, R₄₅ is —CPO)(CH₂)_(q)NH-aryl. In certainembodiments, q is 0. In certain embodiments, the aryl group is phenyl.In certain embodiments, the aryl group is mono-substituted phenyl. Incertain embodiments, the mono-substituted phenyl is further defined asphenyl substituted at the para-position by a halogen. In certainembodiments, R₄₉ is alkoxy, such as methoxy or ethoxy. In certainembodiments R₄₅ is —NHC(O)O-alkyl. In certain embodiments, the alkylgroup of —NHC(O)O-alkyl is a linear heteroatom-unsubstituted alkyl group(e.g., butyl) or a branched heteroatom-unsubstituted alkyl group (e.g.,tert-butyl).

The RAD51 stimulator may, in certain embodiments, be selected from thecompounds shown in FIGS. 8A-8E, or a salt thereof.

It is contemplated that derivatives, metabolites, and prodrugs of thesecompounds may also be used as RAD51 enhancers in some embodiments of theinvention.

In addition, the present invention is contemplated to cover a number ofmethods involving a RAD51 inhibitor, which may decrease, inhibit orreduce a RAD51 activity by or by at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (and any rangederivable therein) compared to RAD51 activity in the absence of theRAD51 inhibitor. Therefore, in some embodiments of the invention, thereare methods for inhibiting RAD51 in a cell comprising providing to thecell an effective amount of a RAD51 small molecule that directlyinhibits RAD51 activity in a cell.

In some embodiments, a RAD51 inhibitor decreases RAD51 filamentformation. In further embodiments, the RAD51 inhibitor does not interactwith a RAD51 DNA binding domain. In still further embodiments, the RAD51inhibitor interferes with RAD51 ATPase activity.

In other embodiments of the invention, there are methods for protectingnon-cancerous cells in a cancer patient comprising administering to thepatient an effective amount of a RAD51 small molecule enhancer prior tobeing exposed to a DNA damaging agent.

Additional embodiments concern methods for sensitizing cancer cells to aDNA damaging agent comprising administering to a cancer patient aneffective amount of a RAD51 small molecule that directly inhibits RAD51activity in a cell. Other embodiments include methods for preventing orreducing DNA damage in a subject comprising administering to the subjectan effective amount of RAD51 enhancer, wherein the enhancer directlyincreases the amount of RAD51 activity.

Other embodiments of the invention include methods for inhibitingconception or the use of compositions of the invention ascontraceptives. Because homologous recombination is required for foamingfunction of eggs and sperm, it is contemplated that RAD51 inhibitors canbe used to reduce fertility of an individual by exposing theindividual's sex cells to an effective amount of a RAD51 inhibitor.

An “effective amount” of a compound or composition, generally, isdefined as that amount sufficient to detectably and repeatedly achievethe stated desired result, for example, to ameliorate, reduce, minimizeor limit the extent of the disease or its symptoms or to increase,stimulate, or promote a desirable physiological response, such ashomologous recombination. More rigorous definitions may apply, includingelimination, eradication or cure of disease.

It is contemplated that in certain embodiments, a cell is a human celland the subject or patient is a human patient. In other embodiments, acell is a mammalian cell and the subject or patient is a mammalianpatient. In some embodiments, a cell is a Drosophila cell and thesubject or patient is a Drosophila patient. It will be understood thatdifferent mammals have their own RAD51 protein that would be a homologof the human protein. In certain other embodiments, the cell is aeukaryotic cell, while in other embodiments, the cell is a prokaryoticcell and a RAD51 protein homolog or analog is the protein that ismodulated. In specific embodiments, a cell may be a sex cell, while inothers, the cell is a somatic cell. In particular embodiments, cellsused in methods of the invention may be from a cell line. In certainembodiments, the cell is a cell from or in any organism describedherein. Moreover, in some embodiments the cell is a cancer cell, whilein other embodiments a cell is non-cancerous or normal. In some cases, acancer cell is resistant to chemotherapy or radiation. Furthermore, itis contemplated that a cell can be in a patient. Additionally, a cellmay be an embryonic stem (ES) cell, such as a murine ES cell, which areused for generating knockout mice. Alternatively, cells may be murinecells that are used for generating a transgenic mouse. Other transgenicanimals can be generated using a particular animals cells in the contextof methods of the invention.

Methods of the invention may involve a RAD51 inhibitor that is a smallmolecular weight compound. In certain embodiments, a RAD51 inhibitor isselected from the group consisting of: (a) a compound of the formula:

or a salt thereof, wherein R₁ is alkyl or a compound of formula (a):

wherein R₃ is alkyl, alkenyl, or alkylamino; Y₁ is NH or O; Z₁ is O orS; and n is 1-5; R₂ is H, alkyl, or aralkyl; and X₁ is a monovalentanion; (b) a compound of the formula:

or a salt thereof, wherein R₄ is H, alkyl, alkoxy or halogen; R₅ is H ornitro; R₆ is H or alkyl; R₇ is H, alkyl, a compound of formula (b):

or a compound of formula (c):

wherein R₉ and R₁₀ are each H or taken together form

R₁₂ is H, alkyl, halogen, or taken together with W₄ forms

W₁-W₄ are each independently CH, CH₂, N, NH, or S; and Z₂-Z₃ are eachindependently S or O; and R₈ is H, alkyl, or halogen; (c) a compound ofthe formula:

or a salt thereof, wherein R₁₄ is H or alkyl; R₁₅ is H, alkyl, ortogether with R₁₆ forms

wherein R₂₃ is H or halogen; R₁₆ is H, alkoxy, hydroxy, or together withR₁₅ forms

wherein R₂₃ is H or halogen; R₁₇ is H or alkyl; R₁₈ is H or togetherwith R₁₉ forms

wherein R₂₄ is H or halogen; R₁₉ is H, halogen, or together with R₁₈forms

wherein R₂₄ is H or halogen; R₂₀ is H, alkyl, or hydroxy; R₂₁ is H ortogether with R₂₂ forms

R₂₂ is H, hydroxy, or together with R₂₁ forms

W₅ and W₆ are each independently CH, CH₂, N, NH, or alkylamino; Y₂ andY₃ are each independently CH, N, N+—O⁻, or SO₂; and m is 0 or 1; (d) acompound of the formula:

or a salt thereof, wherein R₂₅ is H, alkoxy, or nitro; R₂₆ is H ornitro; Y₄ is SO₂ or NH; Y₅ is N or CH; Y₆ is

wherein the point of attachment of Y₆ to the double bond between Y₅ andY₆ is at ring atom A; R₂₇-R₃₂ are each independently H, alkyl, aryl, orhalogen; and Z₄-Z₉ are each independently O or S; and p is 0 or 1; (e) acompound of the formula:

or a salt thereof, wherein R₃₀ is H,

or R₃₀ taken together with R₃₁ forms the fused tricyclic structure

wherein R₃₆ is H or alkyl; and Z₉-Z₁₄ are each independently O or S; R₃₁is H, halogen,

or R₃₁ taken together with R₃₀ forms the fused tricyclic structure

wherein Z₁₄-Z₁₆ are each independently O or S; R₃₇-R₄₀ are eachindependently H, alkyl, or aryl; and X₂ is a monovalent anion; (f) acompound of the formula:

or a salt thereof, wherein R₄₁ and R₄₂ are each independently H orhalogen, or together form

R₄₃ is aryl or

wherein R₄₄ is aryl; and Z₁₉ is O or S; and (g) a compound of formula(VIII):

or a salt thereof, wherein R₅₁ and R₅₂ are each independently H orbromide, and t is 0 or 1, provided that: (i) when R₅₁ is H, then R₅₂ isbromide and t=1; (ii) when R₅₁ is Br and R₅₂ is H, then t=1; and (iii)when R₅₁ and R₅₂ are each bromide, then t=0.

In particular embodiments, a RAD51 inhibitor is selected from thecompounds listed in FIGS. 9A-9K, or a salt thereof.

It is contemplated that derivatives, metabolites, and prodrugs of thesecompounds may also be used as RAD51 inhibitors in some embodiments ofthe invention.

In certain embodiments, the inhibitor is not a nucleotide diphosphate, anucleotide analog, a DNA minor groove binding drug, a xanthine, orxanthine derivative. In particular embodiments, the RAD51 inhibitor doesnot bind to RAD51's DNA binding domain. In certain cases, the RAD51inhibitor interacts with RAD51's ATPase domain.

The small molecules described herein typically contain an aryl group.Accordingly, in certain embodiments, compounds comprising one or morearyl groups are contemplated. The aryl groups may be substituted by anysubstituent known to those of skill in the art (e.g., H, amino, nitro,halo, mercapto, cyano, azido, silyl, hydroxy, alkyl, alkenyl, alkynyl,aryl, aralkyl, alkoxy, alkenoxy, alkynyloxy, aryloxy, acyloxy,alkylamino, alkenylamino, alkynylamino, arylamino, aralkylamino, amido,alkylthio, alkenylthio, alkynylthio, arylthio, aralkylthio, acylthio,alkylsilyl, phosphonate, phosphinate, or any combination thereof).Subsets of these substituent groups at any aryl position are alsocontemplated (e.g., compounds of formula I, II, III, IV, V, VI, VII,VIII, or any combination thereof). In certain embodiments, the smallmolecules are any one or more of the specific chemical compounds whosestructures are shown herein.

In some embodiments, the present invention concerns methods involving anucleic acid to be homologously recombined (exogenous DNA) with a cell'sDNA (endogenous DNA). In some of these embodiments, method involveproviding or administering to a cell a composition comprising a nucleicacid under conditions that promote homologous recombination of all orpart of the nucleic acid with the cell's DNA. In some embodiments, allor part of the exogenous DNA is incorporated into the cell's endogenousDNA. It is contemplated that a cell's endogenous DNA may be chromosomal,episomal, mitochondrial, or nuclear, any of which may be homologouslyrecombined with exogenous DNA.

In certain embodiments, the nucleic acid is in a vector. In specificembodiments the nucleic acid is in a DNA construct. In furtherembodiments, the DNA construct is a plasmid. It is specificallycontemplated that methods of the invention may involve a nucleic acid,all or part of which may undergo homologous recombination with the hostcell's DNA.

In certain embodiments, the nucleic acid is a therapeutic nucleic acidto be introduced into the cell's DNA. A “therapeutic nucleic acid” isdefined herein to refer to a nucleic acid that is known or suspected tobe of benefit in the treatment or prevention of a disease orhealth-related condition. For example, the “therapeutic nucleic acid”may be a nucleic acid that encodes a protein or polypeptide that isknown or suspected to be of benefit in the treatment of a disease orhealth-related condition. Also included in the definition of“therapeutic nucleic acid” is a nucleic acid that transcribes a secondnucleic acid that is known or suspected to be of benefit in thetreatment of a disease or health-related condition (e.g., a DNAtranscribed into ribozyme or siRNA). Alternatively, the “therapeuticnucleic acid” may be one which is known or suspected to provide for atherapeutic benefit without undergoing transcription (e.g., a siRNA or aribozyme).

In other embodiments, the nucleic acid contains one or more alterationsrelative to the cell's endogenous DNA, wherein the alterations will berecombined into the cell's DNA. The alterations may be deletions,insertions, or substitutions of one or more nucleotide residues. Analteration may be a mutation, a wild-type sequence, or a polymorphism.Moreover, in some embodiments of the invention nucleic acids includesequences that allow for their presence to be readily detected orassayed. In certain embodiments, a nucleic acid contains a screenable orselectable marker.

It is contemplated that RAD51 enhancers may be employed in certainembodiments in the following methods: correction of mutations, inductionof specific mutations, insertion of genes under the control of specificpromoters, insertion of promoters to alter regulation of genes, creationof specific chromosome deletions and insertions, creation of specifictranslocations and inversions, increasing the expression of particulargene products, eliminating the expression of particular gene products,introduction of foreign coding regions to endow cells with novelfunctions, introduction of foreign coding regions to test the functionof those regions in a foreign cell including complementation analysis,construction of fusion proteins that facilitate detection andpurification of such proteins with or without their binding partners,construction of combinations of chromosome features to facilitatedetection and measurement of biological processes such as transcription,replication, homologous recombination, and DNA-end-joining.

In certain specific embodiments, methods include “ends-in” DNA vectorconstructs that result in integration of vector circles into thechromosome after linearization in a region of homology. In otherembodiments, methods involve “ends-out” linear constructs that promote“transplacement” of foreign sequences into chromosomes by recombinationof homologous sequences at both ends of the linear construct. Methodsdiscussed above can target a chromosome (previously unaltered or alteredby gene targeting) It is also contemplated that these methods may allowtargeting of an artificial chromosome or episome.

It is contemplated that nucleic acids may be introduced into cells (alsoknown as transfection or transformation) by any method, many of whichare well known to those of skill in the art, including (but not limitedto), electroporation, chemical transformation (calcium phosphate), lipidtransformation, particle bombardment, and microinjection.

In embodiments of the invention, methods may also involve determiningwhether recombination with all or part of the introduced nucleic acidmolecule or sequence has occurred. In specific embodiments, thisinvolves assaying the cells to determine whether the nucleic acid hasbeen incorporated into the cell's nucleic acid.

Methods of the invention can be implemented as treatment for patientswith cancer. The cancer may be any cancer treatable by administration ofa compound described herein. In certain embodiments, a cancer may betreatable using a combination treatment involving a conventional DNAdamaging agent with a RAD51 inhibitor. In some instances, the RAD51inhibitor may be what allows a cancer to be treated with the DNAdamaging agent. Methods of the invention can be implemented with anycancer that may be treated with a DNA damaging agent. For example, thecancer may be breast, prostate, ovarian, brain, melanoma, colorectal,liver, lymphoma, lung, oral, head, neck, spleen, lymph node, smallintestine, large intestine, blood cells, stomach, pancreatic,endometrium, testicle, skin, esophagus, bone marrow, blood, cervical,bladder, Ewing's sarcoma, thyroid, a glioma, and/or gastrointestinal.The invention is applicable to other cancers discussed herein, includingpre-cancers.

In embodiments of the invention, a cancer patient may have been treatedwith or will be treated with a DNA damaging agent. In relatedembodiments, a subject may have been exposed to a DNA damaging agent (asa harmful agent and not as part of a treatment) or be at risk for suchexposure. It is contemplated that in some embodiments of the invention,the DNA damaging agent is an alkylating agent, nitrosourea,anti-metabolite, plant alkaloid, plant extract, or radioisotope. Inspecific embodiments, the DNA damaging agent is actinomycin, amsacrine,anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,daunorubicin, docetaxel, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorethamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel,plicamycin, procarbazine, teniposide, triethylenethiophosphoramide oretoposide (VP16). Any other DNA damaging agent discussed herein may beimplemented in methods of the invention. In certain embodiments, the DNAdamaging agent is radiation.

Methods of the invention may involve multiple administrations of one ormore compounds, compositions, and/or agents. In certain embodiments,cells or a subject are provided with a DNA damaging agent, a RAD51modulator (RAD51 enhancer or inhibitor), and/or a nucleic acid 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 times or more (or any range derivable therein).It is contemplated that compounds, compositions, and/or agents may beformulated in a a pharmaceutically acceptable formulation in certainembodiments of the invention.

Moreover, in methods of the invention the order in which things areprovided to cells or a subject may vary. In some embodiments, a RAD51modulator (RAD51 enhancer and/or RAD51 inhibitor) is provided prior to anucleic acid being provided to a cell or subject. In other embodiments,a RAD51 modulator (RAD51 enhancer and/or RAD51 inhibitor) is providedsimultaneously with a nucleic acid or after the nucleic acid isprovided. It is contemplated that a RAD51 modulator (RAD51 enhancerand/or RAD51 inhibitor) may be provided to a cell or subject within 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48 hours and/or 1, 2, 3, 4, 5, 6, or 7 days(or any range derivable therein) of the cell or subject being providedwith a DNA damaging agent or an exogenous nucleic acid, and vice versa.In certain embodiments, a RAD51 modulator (RAD51 enhancer and/or RAD51inhibitor) is provided before, during, and/or after a DNA damaging agentor an exogenous nucleic acid are provided. In specific embodiments, aRAD51 modulator (RAD51 enhancer and/or RAD51 inhibitor) is providedbefore, during, and/or after gene therapy is provided.

Methods of the invention can involve conferring a therapeutic benefit ona subject. A therapeutic benefit may arise, for example, as a result ofalteration of expression of a particular gene or genes by the nucleicacid. Alteration of expression of a particular gene or genes may beinhibition or augmentation of expression of a particular gene. Inparticular embodiments of the present invention, the therapeutic nucleicacid encodes one or more proteins or polypeptides that can be applied inthe treatment or prevention of a disease or health-related condition ina subject.

A “disease” is defined as a pathological condition of a body part, anorgan, or a system resulting from any cause, such as infection, geneticdefect, or environmental stress. A “health-related condition” is definedherein to refer to a condition of a body part, an organ, or a systemthat may not be pathological, but for which treatment is sought.Examples include conditions for which cosmetic therapy is sought, suchas skin wrinkling, skin blemishes, and the like. The disease can be anydisease, and non-limiting examples include hyperproliferative diseasessuch as cancer and premalignant lesions, wounds, and infections.

“Prevention” and “preventing” are used according to their ordinary andplain meaning to mean “acting before” or such an act. In the context ofa particular disease or health-related condition, those terms refer toadministration or application of an agent, drug, or remedy to a subjector performance of a procedure or modality on a subject for the purposeof blocking the onset of a disease or health-related condition.

The present invention also concerns screening methods for identifying orcharacterizing RAD51 modulators. In some embodiments there arehigh-throughput methods for screening RAD51 modulators comprising: a)combining a RAD51 protein with at least one test compound andfluorescently-labeled oligonucleotides under conditions to promotefilament formation to form a filament formation composition; b)measuring the level of fluorescence polarization (FP). In certainembodiments, a human RAD51 protein is used. Additionally, someembodiments will involve a multi-well plate.

In some screening methods, a filament formation composition may containor contain at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,2.0, 3.0, 4.0, 5.0 μM or more (or any range derivable therein) of RAD51protein. In particular embodiments, RAD51 protein is purified.

Other conditions of the screening method can include a composition thatcomprises calcium ions. It is contemplated that in some embodiments theconcentration of calcium ions in the composition is about, at leastabout, or at most about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 20, 30, 40, 50,60, 70, 80, 90, 100 μM or more (or any range derivable therein).

The present invention also concerns kits and compositions having a RAD51enhancer and a nucleic acid to be introduced into a cell. These kits areexpected to facilitate homologous recombination between the nucleic acidand endogenous DNA.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device and/ormethod being employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claim(s), whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Proposed mechanism for homologous recombinational DNA repair.

FIGS. 2A-2B. FIG. 2A, top: Schematic of protein filament formationmonitored in vitro using a fluorescence polarization assay. FIG. 2A,bottom: Purified RAD51 protein (wild type or mutant F86E) were seriallydiluted. RecA (New England Biolabs) was used as a positive control.Proteins were added to a 5′terminal fluorescein conjugatedoligonucleotide (45 bases of dT) at a 1 μM nucleotide concentration. Themixture was incubated at 37° C. for 30 minutes in 384-well platescontaining a reaction buffer (30 mM Tris/acetate pH 7.5, 10 mMMg²⁺/acetate, 2 mM ATP, 0.1 mM DTT, 0.5 μM BSA). Fluorescencepolarization was measured with a plate reader (filters: excitation=485nm, emission=510 nm). FIG. 2B, top: Varying concentrations of 5′terminalfluorescein conjugated oligonucleotide were used to determine thethreshold RAD51 concentration. FIG. 2B, bottom: Varying the bufferconditions shows that RAD51 filament formation is even more efficient inthe presence of calcium ions.

FIG. 3. (Top) Schematic of AP-peptide fusion assay using RAD51(F86E) asthe protein bait. (Bottom) Wells were coated with various bait proteins(biotinylated RAD51, biotinylated RAD51(F86E), or nothing). Bindingaffinities of two AP-peptide fusions were compared.

FIG. 4. Using an AP-peptide fusion strategy, stepwise truncations of theBRC4 peptide were performed to determine the minimal length sufficientfor binding to RAD51(F86E). The predicted interaction sequence isdisplayed in green italics. A corresponding RAD51 peptide was generated.Point mutations are displayed in red underlined text, including theT1526A and F1524E substitutions in BRC4 and the F86E substitution in theRAD51 fragment (SEQ ID NOS:1-16).

FIG. 5. (Left) RAD51-enhancery compound 45488 (also called compound5253121, or RS-1) (at 20 μM) enhances filament formation. (Right) Thecompound stabilizes RAD51 filaments in the presence of high saltconcentrations.

FIGS. 6A-6B. Schematic of the strand invasion (D-loop) assay. (B) TheD-loop assay was performed using 0.8 μM RAD51 in a buffer that does notsupport joint molecule formation (1 mM ATP, 1 mM MgOAc). Addition of thecompound (45488, also called compound 5253121, or RS-1) lead to aconcentration dependent increase in joint molecule formation (gel onleft, quantification on right).

FIGS. 7A-7C. Cell survival study using a RAD51 enhancer (45488) (alsocalled compound 5253121, or RS-1) at various concentrations and normaldiploid fibroblasts.

FIGS. 8A-8E. Structures of various RAD51 enhancers of the presentinvention.

FIGS. 9A-9K. Structures of various RAD51 inhibitors of the presentinvention.

FIG. 10. Fluorescence polarization was evaluated for a number ofcompounds to identify RAD51 enhancers.

FIG. 11. Fluorescence polarization, which is an assay for filamentformation, was evaluated using different concentrations of RAD51protein.

FIG. 12. Clonogenic survival study using a RAD51 enhancer (45488) (alsocalled compound 5253121, or RS-1) in the presence of radiation atvarious concentrations and chicken DT40 cells.

FIGS. 13A-13E. Fluorescence polarization evaluated for a number ofcompounds to identify RAD51 enhancers.

FIGS. 14A-14C. Fluorescence polarization evaluated for a number ofcompounds to identify RAD51 enhancers.

FIGS. 15A-15C. Results of direct repeat GFP recombination reportersystem assays as performed with certain RAD51 inhibitors.

FIG. 16. D-loop assay using an RAD51 inhibitor.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention overcomes the deficiencies of the prior art byproviding compounds that modulate the activity of a protein involved inhomologous recombinational (HR) DNA repair, RAD51. As discussed above,HR DNA repair confers cellular resistance to ionizing radiation andcertain DNA damaging agents. Pharmacologic manipulation of HR levels maybe used to modulate this resistance. RAD51 is the central proteininvolved in the initiation of HR. Drugs that reduce RAD51 function couldpotentially be used to inhibit HR in cancer cells, thereby overcomingthe resistance of malignant tumors to common oncologic therapies.Conversely, RAD51 enhancing agents could serve to transiently increaseHR and/or bypass the dependence of HR on RAD51 mediator proteins. Suchagents could potentially elevate cellular resistance to DNA damagingagents, thereby increasing cell survival and potentially reducingmutagenesis. This type of agent could be used to protect normal tissuesin patients receiving oncologic therapies. Such agents also could serveas personal protection in the event of a nuclear disaster or a ‘dirtybomb.’

A targeted approach that focuses on HR is particularly appealing, sinceseveral studies have suggested that HR inhibition preferentiallysensitizes tumor cells relative to normal cells (Russell et al., 2003;Ito et al., 2005). In one such study, Gleevec-mediated inhibition ofRAD51 expression resulted in an elevated radio-sensitivity for gliomacancer cell lines, but had no significant effect on normal humanfibroblasts (Russell et al., 2003). Similarly, Ito et. al. blocked RAD51expression with siRNA techniques in various human cancer cell lines. Thesensitivity to cisplatin was enhanced in all of the human cancer celllines tested, but not in normal human fibroblasts (Ito et al., 2005).Therefore, a strategy for pharmacologic inhibition of HR holds promisefor improving the therapeutic ratios of existing oncology treatments.

A. RAD51 Protein

RAD51 filament formation is a well accepted critical step in theinitiation of HR repair. Biochemical studies have shown that RAD51protein assembles into filaments readily on sites of single stranded DNA(ssDNA). In vitro filament formation is magnesium and ATP dependent, andrequires a concentration of RAD51 protein of approximately 250 nM. Thisreaction also demonstrates cooperativity, such that a threshold level ofRAD51 binding to ssDNA will stimulate further filament formation((Zaitseva et al., 1999; Shinohara et al., 1992). One mechanism by whichcells can up-regulate filament formation is over-expression of RAD51protein. RAD51 is over-expressed in a number of human cancer cell lines(Raderschall et al., 2002; Russell et al., 2003; Hansen et al., 2003)and human tumors (Maacke et al., 2000a; Maacke et al., 2000b; Han etal., 2002; Henning and Sturzbecher, 2003; Yoshikawa et al., 2000; Qiaoet al., 2005). Fluorescence in situ hybridization (FISH) experimentshave shown that the RAD51 gene is not amplified. Other experiments havedemonstrated that RAD51 protein half-life is normal in tumor cells.Taken together, this suggests that the increased protein levels resultfrom transcriptional up-regulation (Raderschall et al., 2002). There arealso data to suggest that RAD51 over-expression can compensate for lossof RAD51 paralog proteins, thus by-passing the need for a mediatorprotein that would have otherwise been rate limiting (Takata et al.,2001). RAD51 over-expression is particularly dramatic in the case ofpancreatic cancer. Han et. al. (2002) performed a cDNA microarrayanalysis comparing pancreatic cancer cells lines to normal pancreaticcells; RAD51 was among the 30 most over-expressed genes in thisanalysis. This result was confirmed with an immunohistochemical (IHC)analysis showing strong RAD51 staining in 71.8% of malignant pancreatictumors in humans (Han et al., 2002). A similar study of 47 humanpancreatic tumor tissue specimens showed RAD51 overexpression in 66% oftumors (Maacke et al., 2000b). In fact, RAD51 overexpression is so greatthat 7% of pancreatic cancer patients generate auto-antibodies to RAD51,which can be detected in their sera (Maacke et al., 2002). A functionalanalysis using a system of inducible RAD51 overexpression in pancreaticcells directly showed that overexpression confers resistance to DSB's(Maacke et al., 2000b). A growing body of literature agrees with thisfinding, suggesting that high RAD51 protein expression levels canmodulate the resistance of cancer cells to IR (ionizing radiation) andsome chemotherapeutic drugs.

B. Homologous Recombination and BRCA1 Mutations

The breast cancer susceptibility gene BRCA2 plays an important role inHR (Moynahan et al., 2001). It has been speculated that BRCA2 proteineither chaperones RAD51 monomers, that it serves as mediator for RAD51filament assembly at sites of DNA damage, and/or that it acts tostabilize existing filaments. A recent report suggested that RAD51directly interacts with a C-terminal region of BRCA2 protein (Esashi etal., 2005). RAD51 is also known to directly bind six of the eightconserved BRC repeats within human BRCA2 (Chen et al., 1998; Wong etal., 1997). Crystal structure data have been generated using a fusionprotein containing BRC4 covalently linked to the C-terminal core domainof RAD51. This and other studies have demonstrated that BRC repeatsshare a conserved motif that is thought to mimic a primary interfaceused in RAD51 polymerization (Shin et al., 2003; Pellegrini et al.,2002). Various BRC peptides have been shown capable of blockingself-association by RAD51 monomers, inhibiting RAD51 filament formation,and sensitizing cells to DNA damage (Chen et al., 1999; Davies et al.,2001; Yuan et al., 1999). For example Chen et. al. reported thatconditional expression of wild-type BRC4 resulted in hypersensitivity toirradiation and an inability to form radiation-induced RAD51 foci inbreast cancer cells (Chen et al., 1999).

Preliminary tumor studies by the present inventors focused on HR bymodulating the RAD51 paralog protein XRCC3. Using xenograft tumors grownin nude mice, cisplatin treatment was found to produce regression andcure of HR-deficient xenograft tumors using a treatment schedule thathas little effect of HR-competent tumors. Similarly, the HR-deficienttumors were sensitive to relatively low doses of ionizing radiation,compared to HR-competent tumors. Other clinical studies suggest that,like XRCC3-deficient tumors, HR-deficient human tumors (due toBRCA1-mutation) are especially susceptible to DNA damagingchemotherapeutic agents (Porter et al., 1994; Rubin et al., 1996).Tanaka and colleagues found that in a group ovarian cancer patientsreceiving ciplatin treatment, those defective for BRCA1 had a five-yearsurvival of 79% while patients in the control group had a five-yearsurvival of only 30% (Alda et al., 1998). Similarly, Marcus et al.(1996) found that BRCA1-defective women with breast cancer hadrelatively favorable outcomes, despite having tumors with higher gradeand higher S-phase fraction. Thus, deficiencies of HR may sensitizetumor cells to some chemotherapy drugs and IR, providing evidence thatpharmacologic inhibitors of HR are likely to improve the clinicalefficacy of cancer treatments.

C. Cancer and DNA Damaging Agents

In certain embodiments, the invention is applicable to the treatment ofcancer insofar as such treatments may involve DNA damaging agents.

Cancer cells that may be treated by methods and compositions of theinvention also include cells from the bladder, blood, bone, bone marrow,brain, breast, colon, esophagus, gastrointestine, gum, head, kidney,liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis,tongue, or uterus. In addition, the cancer may specifically be of thefollowing histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; androblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The term “DNA damaging agent” refers to any agent that directly orindirectly damages DNA for which homologous recombination could repairthe damage. Specific examples of DNA-damaging agents include alkylatingagents, nitrosoureas, anti-metabolites, plant alkaloids, plant extractsand radioisotopes. Specific examples of agents also include DNA-damagingdrugs, for example, 5-fluorouracil (5-FU), capecitabine, S-1 (Tegafur,5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil,arabinosyl cytosine (ara-C), 5-azacytidine (5-AC),2′,2′-difluoro-2′-deoxycytidine (dFdC), purine antimetabolites(mercaptopurine, azathiopurine, thioguanine), gemcitabine hydrochlorine(Gemzar), pentostatin, allopurinol, 2-fluoro-arabinosyl-adenine(2F-ara-A), hydroxyurea, sulfur mustard (bischloroetyhylsulfide),mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide,thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, alkylsulfonate (busulfan), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU),procarbazine, decarbazine, rebeccamycin, anthracyclins such asdoxorubicin (adriamycin; ADR), daunorubicin (Cerubicine), idarubicin(Idamycin) and epirubicin (Ellence), anthracyclin analogs such asmitoxantrone, actinimycin D, non-intercalating topoisomerase inhibitorssuch as epipodophyllotoxins (etoposide or VP16, teniposide or VM-26),podophylotoxin, bleomycin (Bleo), pepleomycin, compounds that formadducts with nucleic acid including platinum derivatives, e.g.,cisplatin (CDDP), trans analog of cisplatin, carboplatin, iproplatin,tetraplatin and oxaliplatin, as well as camptothecin, topotecan,irinotecan (CPT-11), and SN-38. Specific examples of nucleic aciddamaging treatments include radiation e.g., ultraviolet (UV), infrared(IR), or α-, β-, or γ-radiation, as well as environmental shock, e.g.,hyperthermia. One of skill in the art can identify and use otherDNA-damaging agents and treatments.

D. Chemical Definitions

As used herein, a “small molecule” refers to an organic compound that iseither synthesized via conventional organic chemistry methods (e.g., ina laboratory) or found in nature. Typically, a small molecule ischaracterized in that it contains several carbon-carbon bonds, and has amolecular weight of less than about 1500 grams/mole. In certainembodiments, small molecules are less than about 1000 grams/mole. Incertain embodiments, small molecules are less than about 550 grams/mole.In certain embodiments, small molecules are between about 200 and about550 grams/mole. In certain embodiments, small molecules exclude peptides(e.g., compounds comprising 2 or more amino acids joined by a peptidylbond). In certain embodiments, small molecules exclude nucleic acids.

As used herein, the term “amino” means —NH₂; the term “nitro” means—NO₂; the term “halo” or “halogen” designates —F, —Cl, —Br or —I; theterm “mercapto” means —SH; the term “cyano” means —CN; the term “azido”means —N₃; the term “silyl” means —SiH₃, and the term “hydroxy” means—OH. In certain embodiments, a halogen may be —Br or —I.

As used herein, a “monovalent anion” refers to anions of a −1 charge.Such anions are well-known to those of skill in the art. Non-limitingexamples of monovalent anions include halides (e.g., F⁻, Cl⁻, Br⁻ andI⁻), NO₂ ⁻, NO₃ ⁻, hydroxide (OH⁻) and azide (N₃ ⁻).

As used herein, the structure

indicates that the bond may be a single bond or a double bond. Those ofskill in the chemical arts understand that in certain circumstances, adouble bond between two particular atoms is chemically feasible and incertain circumstances, a double bond is not. The present inventiontherefore contemplates that a double bond may be formed only whenchemically feasible.

The term “alkyl” includes straight-chain alkyl, branched-chain alkyl,cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl,heteroatom-substituted alkyl, heteroatom-unsubstituted C_(n)-alkyl, andheteroatom-substituted C_(n)-alkyl. In certain embodiments, lower alkylsare contemplated. The term “lower alkyl” refers to alkyls of 1-6 carbonatoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term“heteroatom-unsubstituted C_(n)-alkyl” refers to a radical, having alinear or branched, cyclic or acyclic structure, further having nocarbon-carbon double or triple bonds, further having a total of n carbonatoms, all of which are nonaromatic, 3 or more hydrogen atoms, and noheteroatoms. For example, a heteroatom-unsubstituted C₁-C₁₀-alkyl has 1to 10 carbon atoms. The groups, —CH₃ (Me), —CH₂CH₃ (Et), —CH₂CH₂CH₃(n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl), —CH₂CH₂CH₂CH₃(n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂ (iso-butyl), —C(CH₃)₃(tent-butyl), —CH₂C(CH₃)₃ (neo-pentyl), cyclobutyl, cyclopentyl, andcyclohexyl, are all non-limiting examples of heteroatom-unsubstitutedalkyl groups. The term “heteroatom-substituted C_(n)-alkyl” refers to aradical, having a single saturated carbon atom as the point ofattachment, no carbon-carbon double or triple bonds, further having alinear or branched, cyclic or acyclic structure, further having a totalof n carbon atoms, all of which are nonaromatic, 0, 1, or more than onehydrogen atom, at least one heteroatom, wherein each heteroatom isindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. For example, a heteroatom-substituted C₁-C₁₀-alkyl has 1to 10 carbon atoms. The following groups are all non-limiting examplesof heteroatom-substituted alkyl groups: trifluoromethyl, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂OH, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, CH₂CH₂OC(O)CH₃, —CH₂CH₂NHCO₂C(CH₃)₃,and —CH₂Si(CH₃)₃.

The term “alkenyl” includes straight-chain alkenyl, branched-chainalkenyl, cycloalkenyl, cyclic alkenyl, heteroatom-unsubstituted alkenyl,heteroatom-substituted alkenyl, heteroatom-unsubstituted C_(n)-alkenyl,and heteroatom-substituted C_(n)-alkenyl. In certain embodiments, loweralkenyls are contemplated. The term “lower alkenyl” refers to alkenylsof 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term“heteroatom-unsubstituted C_(n)-alkenyl” refers to a radical, having alinear or branched, cyclic or acyclic structure, further having at leastone nonaromatic carbon-carbon double bond, but no carbon-carbon triplebonds, a total of n carbon atoms, three or more hydrogen atoms, and noheteroatoms. For example, a heteroatom-unsubstituted C₂-C₁₀-alkenyl has2 to 10 carbon atoms. Heteroatom-unsubstituted alkenyl groups include:—CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl),—CH₂CH═CHCH₃, and —CH═CH—C₆H₅. The term “heteroatom-substitutedC_(n)-alkenyl” refers to a radical, having a single nonaromatic carbonatom as the point of attachment and at least one nonaromaticcarbon-carbon double bond, but no carbon-carbon triple bonds, furtherhaving a linear or branched, cyclic or acyclic structure, further havinga total of n carbon atoms, 0, 1, or more than one hydrogen atom, and atleast one heteroatom, wherein each heteroatom is independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Forexample, a heteroatom-substituted C₂-C₁₀-alkenyl has 2 to 10 carbonatoms. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, are non-limitingexamples of heteroatom-substituted alkenyl groups.

The term “aryl” includes heteroatom-unsubstituted aryl,heteroatom-substituted aryl, heteroatom-unsubstituted C_(n)-aryl,heteroatom-substituted C_(n)-aryl, heteroaryl, heterocyclic aryl groups,carbocyclic aryl groups, biaryl groups, and single-valent radicalsderived from polycyclic fused hydrocarbons (PAHs). The term“heteroatom-unsubstituted C_(n)-aryl” refers to a radical, having asingle carbon atom as a point of attachment, wherein the carbon atom ispart of an aromatic ring structure containing only carbon atoms, furtherhaving a total of n carbon atoms, 5 or more hydrogen atoms, and noheteroatoms. For example, a heteroatom-unsubstituted C₆-C₁₀-aryl has 6to 10 carbon atoms. Non-limiting examples of heteroatom-unsubstitutedaryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl,—C₆H₄CH₂CH₃, —C₆H₄CH₂CH₂CH₃, —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂,—C₆H₃(CH₃)CH₂CH₃, —C₆H₄CH═CH₂, —C₆H₄CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃,naphthyl, and the radical derived from biphenyl. The term“heteroatom-substituted C_(n)-aryl” refers to a radical, having either asingle aromatic carbon atom or a single aromatic heteroatom as the pointof attachment, further having a total of n carbon atoms, at least onehydrogen atom, and at least one heteroatom, further wherein eachheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstitutedC₁-C₁₀-heteroaryl has 1 to 10 carbon atoms. Non-limiting examples ofheteroatom-substituted aryl groups include the groups: —C₆H₄F, —C₆H₄Cl,—C₆H₄Br, —C₆H₄I, —C₆H₄OH, —C₆H₄OCH₃, —C₆H₄OCH₂CH₃, —C₆H₄OC(O)CH₃,—C₆H₄NH₂, —C₆H₄NHCH₃, —C₆H₄N(CH₃)₂, —C₆H₄CH₂OH, —C₆H₄CH₂OC(O)CH₃,—C₆H₄CH₂NH₂, —C₆H₄CF₃, —C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO, —C₆H₄C(O)CH₃,—C₆H₄C(O)C₆H₅, —C₆H₄CO₂H, —C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃,—C₆H₄CON(CH₃)₂, furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl,pyrazinyl, quinolyl, indolyl, and imidazoyl. In certain embodiments,heteroatom-substituted aryl groups are contemplated. In certainembodiments, heteroatom-unsubstituted aryl groups are contemplate. Incertain embodiments, an aryl group may be mono-, di-, tri-, tetra- orpenta-substituted with one or more heteroatom-containing substitutents.

The term “aralkyl” includes heteroatom-unsubstituted aralkyl,heteroatom-substituted aralkyl, heteroatom-unsubstituted C_(n)-aralkyl,heteroatom-substituted C_(n)-aralkyl, heteroaralkyl, and heterocyclicaralkyl groups. In certain embodiments, lower aralkyls are contemplated.The term “lower aralkyl” refers to aralkyls of 7-12 carbon atoms (thatis, 7, 8, 9, 10, 11 or 12 carbon atoms). The term“heteroatom-unsubstituted C_(n)-aralkyl” refers to a radical, having asingle saturated carbon atom as the point of attachment, further havinga total of n carbon atoms, wherein at least 6 of the carbon atoms forman aromatic ring structure containing only carbon atoms, 7 or morehydrogen atoms, and no heteroatoms. For example, aheteroatom-unsubstituted C₇-C₁₀-aralkyl has 7 to 10 carbon atoms.Non-limiting examples of heteroatom-unsubstituted aralkyls are:phenylmethyl (benzyl, Bn) and phenylethyl. The term“heteroatom-substituted C_(n)-aralkyl” refers to a radical, having asingle saturated carbon atom as the point of attachment, further havinga total of n carbon atoms, 0, 1, or more than one hydrogen atom, and atleast one heteroatom, wherein at least one of the carbon atoms isincorporated an aromatic ring structures, further wherein eachheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substitutedC₂-C₁₀-heteroaralkyl has 2 to 10 carbon atoms.

The term “acyl” includes straight-chain acyl, branched-chain acyl,cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl,heteroatom-substituted acyl, heteroatom-unsubstituted C_(n)-acyl,heteroatom-substituted C_(n)-acyl, alkylcarbonyl, alkoxycarbonyl andaminocarbonyl groups. In certain embodiments, lower acyls arecontemplated. The term “lower acyl” refers to acyls of 1-6 carbon atoms(that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term“heteroatom-unsubstituted C_(n)-acyl” refers to a radical, having asingle carbon atom of a carbonyl group as the point of attachment,further having a linear or branched, cyclic or acyclic structure,further having a total of n carbon atoms, 1 or more hydrogen atoms, atotal of one oxygen atom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₁-C₁₀-acyl has 1 to 10 carbon atoms. Thegroups, —CHO, —C(O)CH₃, —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂,—C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)C₆H₄CH₂CH₃, and—COC₆H₃(CH₃)₂, are non-limiting examples of heteroatom-unsubstitutedacyl groups. The term “heteroatom-substituted C_(n)-acyl” refers to aradical, having a single carbon atom as the point of attachment, thecarbon atom being part of a carbonyl group, further having a linear orbranched, cyclic or acyclic structure, further having a total of ncarbon atoms, 0, 1, or more than one hydrogen atom, at least oneadditional heteroatom, in addition to the oxygen of the carbonyl group,wherein each additional heteroatom is independently selected from thegroup consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₁-C₁₀-acyl has 1 to 10 carbon atoms. The groups,—C(O)CH₂CF₃, —CO₂H, —CO₂ ⁻, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃,—CO₂CH(CH₃)₂, —CO₂CH(CH₂)₂, —C(O)NH₂ (carbamoyl), —C(O)NHCH₃,—C(O)NHCH₂CH₃, —CONHCH(CH₃)₂, —CONHCH(CH₂)₂, —CON(CH₃)₂, and—CONHCH₂CF₃, are non-limiting examples of heteroatom-substituted acylgroups.

The term “alkoxy” includes straight-chain alkoxy, branched-chain alkoxy,cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy,heteroatom-substituted alkoxy, heteroatom-unsubstituted C_(n)-alkoxy,and heteroatom-substituted C_(n)-alkoxy. In certain embodiments, loweralkoxys are contemplated. The term “lower alkoxy” refers to alkoxys of1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term“heteroatom-unsubstituted C_(n)-alkoxy” refers to a group, having thestructure —OR, in which R is a heteroatom-unsubstituted C_(n)-alkyl, asthat term is defined above. Heteroatom-unsubstituted alkoxy groupsinclude: —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃, —OCH(CH₃)₂, and —OCH(CH₂)₂. Theterm “heteroatom-substituted C_(n)-alkoxy” refers to a group, having thestructure —OR, in which R is a heteroatom-substituted C_(n)-alkyl, asthat term is defined above. For example, —OCH₂CF₃ is aheteroatom-substituted alkoxy group.

The term “alkenyloxy” includes straight-chain alkenyloxy, branched-chainalkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstitutedalkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstitutedC_(n)-alkenyloxy, and heteroatom-substituted C_(n)-alkenyloxy. The term“heteroatom-unsubstituted C_(n)-alkenyloxy” refers to a group, havingthe structure —OR, in which R is a heteroatom-unsubstitutedC_(n)-alkenyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkenyloxy” refers to a group, having thestructure —OR, in which R is a heteroatom-substituted C_(n)-alkenyl, asthat term is defined above.

The term “alkynyloxy” includes straight-chain alkynyloxy, branched-chainalkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstitutedalkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstitutedC_(n)-alkynyloxy, and heteroatom-substituted C_(n)-alkynyloxy. The term“heteroatom-unsubstituted C_(n)-alkynyloxy” refers to a group, havingthe structure —OR, in which R is a heteroatom-unsubstitutedC_(n)-alkynyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkynyloxy” refers to a group, having thestructure —OR, in which R is a heteroatom-substituted C_(n)-alkynyl, asthat term is defined above.

The term “aryloxy” includes heteroatom-unsubstituted aryloxy,heteroatom-substituted aryloxy, heteroatom-unsubstituted C_(n)-aryloxy,heteroatom-substituted C_(n)-aryloxy, heteroaryloxy, and heterocyclicaryloxy groups. The term “heteroatom-unsubstituted C_(n)-aryloxy” refersto a group, having the structure —OAr, in which Ar is aheteroatom-unsubstituted C_(n)-aryl, as that term is defined above. Anon-limiting example of a heteroatom-unsubstituted aryloxy group is—OC₆H₅. The term “heteroatom-substituted C_(n)-aryloxy” refers to agroup, having the structure —OAr, in which Ar is aheteroatom-substituted C_(n)-aryl, as that term is defined above.

The term “aralkyloxy” includes heteroatom-unsubstituted aralkyloxy,heteroatom-substituted aralkyloxy, heteroatom-unsubstitutedC_(n)-aralkyloxy, heteroatom-substituted C_(n)-aralkyloxy,heteroaralkyloxy, and heterocyclic aralkyloxy groups. The term“heteroatom-unsubstituted C_(n)-aralkyloxy” refers to a group, havingthe structure —OAr, in which Ar is a heteroatom-unsubstitutedC_(n)-aralkyl, as that term is defined above. The term“heteroatom-substituted C_(n)-aralkyloxy” refers to a group, having thestructure —OAr, in which Ar is a heteroatom-substituted C_(n)-aralkyl,as that term is defined above.

The term “acyloxy” includes straight-chain acyloxy, branched-chainacyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy,heteroatom-substituted acyloxy, heteroatom-unsubstituted C_(n)-acyloxy,heteroatom-substituted C_(n)-acyloxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term“heteroatom-unsubstituted C_(n)-acyloxy” refers to a group, having thestructure —OAc, in which Ac is a heteroatom-unsubstituted C_(n)-acyl, asthat term is defined above. For example, —OC(O)CH₃ is a non-limitingexample of a heteroatom-unsubstituted acyloxy group. The term“heteroatom-substituted C_(n)-acyloxy” refers to a group, having thestructure —OAc, in which Ac is a heteroatom-substituted C_(n)-acyl, asthat term is defined above. For example, —OC(O)OCH₃ and —OC(O)NHCH₃ arenon-limiting examples of heteroatom-unsubstituted acyloxy groups.

The term “alkylamino” includes straight-chain alkylamino, branched-chainalkylamino, cycloalkylamino, cyclic alkylamino, heteroatom-unsubstitutedalkylamino, heteroatom-substituted alkylamino, heteroatom-unsubstitutedC_(n)-alkylamino, and heteroatom-substituted C_(n)-alkylamino. The term“heteroatom-unsubstituted C_(n)-alkylamino” refers to a radical, havinga single nitrogen atom as the point of attachment, further having one ortwo saturated carbon atoms attached to the nitrogen atom, further havinga linear or branched, cyclic or acyclic structure, containing a total ofn carbon atoms, all of which are nonaromatic, 4 or more hydrogen atoms,a total of 1 nitrogen atom, and no additional heteroatoms. For example,a heteroatom-unsubstituted C₁-C₁₀-alkylamino has 1 to 10 carbon atoms.The term “heteroatom-unsubstituted C_(n)-alkylamino” includes groups,having the structure —NHR, in which R is a heteroatom-unsubstitutedC_(n)-alkyl, as that term is defined above. A heteroatom-unsubstitutedalkylamino group would include —NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃,—NHCH(CH₃)₂, —NHCH(CH₂)₂, —NHCH₂CH₂CH₂CH₃, —NHCH(CH₃)CH₂CH₃,—NHCH₂CH(CH₃)₂, —NHC(CH₃)₃, —N(CH₃)₂, —N(CH₃)CH₂CH₃, —N(CH₂CH₃)₂,N-pyrrolidinyl, and N-piperidinyl. The term “heteroatom-substitutedC_(n)-alkylamino” refers to a radical, having a single nitrogen atom asthe point of attachment, further having one or two saturated carbonatoms attached to the nitrogen atom, no carbon-carbon double or triplebonds, further having a linear or branched, cyclic or acyclic structure,further having a total of n carbon atoms, all of which are nonaromatic,0, 1, or more than one hydrogen atom, and at least one additionalheteroatom, that is, in addition to the nitrogen atom at the point ofattachment, wherein each additional heteroatom is independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Forexample, a heteroatom-substituted C₁-C₁₀-alkylamino has 1 to 10 carbonatoms. The term “heteroatom-substituted C_(n)-alkylamino” includesgroups, having the structure —NHR, in which R is aheteroatom-substituted C_(n)-alkyl, as that term is defined above.

The term “alkenylamino” includes straight-chain alkenylamino,branched-chain alkenylamino, cycloalkenylamino, cyclic alkenylamino,heteroatom-unsubstituted alkenylamino, heteroatom-substitutedalkenylamino, heteroatom-unsubstituted C_(n)-alkenylamino,heteroatom-substituted C_(n)-alkenylamino, dialkenylamino, andalkyl(alkenyl)amino groups. The term “heteroatom-unsubstitutedC_(n)-alkenylamino” refers to a radical, having a single nitrogen atomas the point of attachment, further having one or two carbon atomsattached to the nitrogen atom, further having a linear or branched,cyclic or acyclic structure, containing at least one nonaromaticcarbon-carbon double bond, a total of n carbon atoms, 4 or more hydrogenatoms, a total of one nitrogen atom, and no additional heteroatoms. Forexample, a heteroatom-unsubstituted C₂-C₁₀-alkenylamino has 2 to 10carbon atoms. The term “heteroatom-unsubstituted C_(n)-alkenylamino”includes groups, having the structure —NHR, in which R is aheteroatom-unsubstituted C_(n)-alkenyl, as that term is defined above.The term “heteroatom-substituted C_(n)-alkenylamino” refers to aradical, having a single nitrogen atom as the point of attachment and atleast one nonaromatic carbon-carbon double bond, but no carbon-carbontriple bonds, further having one or two carbon atoms attached to thenitrogen atom, further having a linear or branched, cyclic or acyclicstructure, further having a total of n carbon atoms, 0, 1, or more thanone hydrogen atom, and at least one additional heteroatom, that is, inaddition to the nitrogen atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₂-C₁₀-alkenylamino has 2 to 10 carbon atoms. Theterm “heteroatom-substituted C_(n)-alkenylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-substitutedC_(n)-alkenyl, as that term is defined above.

The term “alkynylamino” includes straight-chain alkynylamino,branched-chain alkynylamino, cycloalkynylamino, cyclic alkynylamino,heteroatom-unsubstituted alkynylamino, heteroatom-substitutedalkynylamino, heteroatom-unsubstituted C_(n)-alkynylamino,heteroatom-substituted C_(n)-alkynylamino, dialkynylamino,alkyl(alkynyl)amino, and alkenyl(alkynyl)amino groups. The term“heteroatom-unsubstituted C_(n)-alkynylamino” refers to a radical,having a single nitrogen atom as the point of attachment, further havingone or two carbon atoms attached to the nitrogen atom, further having alinear or branched, cyclic or acyclic structure, containing at least onecarbon-carbon triple bond, a total of n carbon atoms, at least onehydrogen atoms, a total of one nitrogen atom, and no additionalheteroatoms. For example, a heteroatom-unsubstituted C₂-C₁₀-alkynylaminohas 2 to 10 carbon atoms. The term “heteroatom-unsubstitutedC_(n)-alkynylamino” includes groups, having the structure —NHR, in whichR is a heteroatom-unsubstituted C_(n)-alkynyl, as that term is definedabove. The term “heteroatom-substituted C_(n)-alkynylamino” refers to aradical, having a single nitrogen atom as the point of attachment,further having one or two carbon atoms attached to the nitrogen atom,further having at least one nonaromatic carbon-carbon triple bond,further having a linear or branched, cyclic or acyclic structure, andfurther having a total of n carbon atoms, 0, 1, or more than onehydrogen atom, and at least one additional heteroatom, that is, inaddition to the nitrogen atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₂-C₁₀-alkynylamino has 2 to 10 carbon atoms. Theterm “heteroatom-substituted C_(n)-alkynylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-substitutedC_(n)-alkynyl, as that term is defined above.

The term “arylamino” includes heteroatom-unsubstituted arylamino,heteroatom-substituted arylamino, heteroatom-unsubstitutedC_(n)-arylamino, heteroatom-substituted C_(n)-arylamino,heteroarylamino, heterocyclic arylamino, and alkyl(aryl)amino groups.The term “heteroatom-unsubstituted C_(n)-arylamino” refers to a radical,having a single nitrogen atom as the point of attachment, further havingat least one aromatic ring structure attached to the nitrogen atom,wherein the aromatic ring structure contains only carbon atoms, furtherhaving a total of n carbon atoms, 6 or more hydrogen atoms, a total ofone nitrogen atom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₆-C₁₀-arylamino has 6 to 10 carbon atoms. Theterm “heteroatom-unsubstituted C_(n)-arylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-unsubstituted C_(n)-aryl,as that term is defined above. The term “heteroatom-substitutedC_(n)-arylamino” refers to a radical, having a single nitrogen atom asthe point of attachment, further having a total of n carbon atoms, atleast one hydrogen atom, at least one additional heteroatoms, that is,in addition to the nitrogen atom at the point of attachment, wherein atleast one of the carbon atoms is incorporated into one or more aromaticring structures, further wherein each additional heteroatom isindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. For example, a heteroatom-substituted C₆-C₁₀-arylamino has6 to 10 carbon atoms. The term “heteroatom-substituted C_(n)-arylamino”includes groups, having the structure —NHR, in which R is aheteroatom-substituted C_(n)-aryl, as that term is defined above.

The term “aralkylamino” includes heteroatom-unsubstituted aralkylamino,heteroatom-substituted aralkylamino, heteroatom-unsubstitutedC_(n)-aralkylamino, heteroatom-substituted C_(n)-aralkylamino,heteroaralkylamino, heterocyclic aralkylamino groups, and diaralkylaminogroups. The term “heteroatom-unsubstituted C_(n)-aralkylamino” refers toa radical, having a single nitrogen atom as the point of attachment,further having one or two saturated carbon atoms attached to thenitrogen atom, further having a total of n carbon atoms, wherein atleast 6 of the carbon atoms form an aromatic ring structure containingonly carbon atoms, 8 or more hydrogen atoms, a total of one nitrogenatom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₇-C₁₀-aralkylamino has 7 to 10 carbon atoms.The term “heteroatom-unsubstituted C_(n)-aralkylamino” includes groups,having the structure —NHR, in which R is a heteroatom-unsubstitutedC_(n)-aralkyl, as that term is defined above. The term“heteroatom-substituted C_(n)-aralkylamino” refers to a radical, havinga single nitrogen atom as the point of attachment, further having atleast one or two saturated carbon atoms attached to the nitrogen atom,further having a total of n carbon atoms, 0, 1, or more than onehydrogen atom, at least one additional heteroatom, that is, in additionto the nitrogen atom at the point of attachment, wherein at least one ofthe carbon atom incorporated into an aromatic ring, further wherein eachheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substitutedC₇-C₁₀-aralkylamino has 7 to 10 carbon atoms. The term“heteroatom-substituted C_(n)-aralkylamino” includes groups, having thestructure —NHR, in which R is a heteroatom-substituted C_(n)-aralkyl, asthat term is defined above.

The term “amido” includes straight-chain amido, branched-chain amido,cycloamido, cyclic amido, heteroatom-unsubstituted amido,heteroatom-substituted amido, heteroatom-unsubstituted C_(n)-amido,heteroatom-substituted C_(n)-amido, alkylcarbonylamino,arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, acylamino,alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups. Theterm “heteroatom-unsubstituted C_(n)-amido” refers to a radical, havinga single nitrogen atom as the point of attachment, further having acarbonyl group attached via its carbon atom to the nitrogen atom,further having a linear or branched, cyclic or acyclic structure,further having a total of n carbon atoms, 1 or more hydrogen atoms, atotal of one oxygen atom, a total of one nitrogen atom, and noadditional heteroatoms. For example, a heteroatom-unsubstitutedC₁-C₁₀-amido has 1 to 10 carbon atoms. The term“heteroatom-unsubstituted C_(n)-amido” includes groups, having thestructure —NHR, in which R is a heteroatom-unsubstituted C_(n)-acyl, asthat term is defined above. The group, —NHC(O)CH₃, is a non-limitingexample of a heteroatom-unsubstituted amido group. The term“heteroatom-substituted C_(n)-amido” refers to a radical, having asingle nitrogen atom as the point of attachment, further having acarbonyl group attached via its carbon atom to the nitrogen atom,further having a linear or branched, cyclic or acyclic structure,further having a total of n aromatic or nonaromatic carbon atoms, 0, 1,or more than one hydrogen atom, at least one additional heteroatom inaddition to the oxygen of the carbonyl group, wherein each additionalheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substitutedC₁-C₁₀-amido has 1 to 10 carbon atoms. The term “heteroatom-substitutedC_(n)-amido” includes groups, having the structure —NHR, in which R is aheteroatom-unsubstituted C_(n)-acyl, as that term is defined above. Thegroup, —NHCO₂CH₃, is a non-limiting example of a heteroatom-substitutedamido group.

The term “alkylthio” includes straight-chain alkylthio, branched-chainalkylthio, cycloalkylthio, cyclic alkylthio, heteroatom-unsubstitutedalkylthio, heteroatom-substituted alkylthio, heteroatom-unsubstitutedC_(n)-alkylthio, and heteroatom-substituted C_(n)-alkylthio. The term“heteroatom-unsubstituted C_(n)-alkylthio” refers to a group, having thestructure —SR, in which R is a heteroatom-unsubstituted C_(n)-alkyl, asthat term is defined above. The group, —SCH₃, is an example of aheteroatom-unsubstituted alkylthio group. The term“heteroatom-substituted C_(n)-alkylthio” refers to a group, having thestructure —SR, in which R is a heteroatom-substituted C_(n)-alkyl, asthat term is defined above.

The term “alkenylthio” includes straight-chain alkenylthio,branched-chain alkenylthio, cycloalkenylthio, cyclic alkenylthio,heteroatom-unsubstituted alkenylthio, heteroatom-substitutedalkenylthio, heteroatom-unsubstituted C_(n)-alkenylthio, andheteroatom-substituted C_(n)-alkenylthio. The term“heteroatom-unsubstituted C_(n)-alkenylthio” refers to a group, havingthe structure —SR, in which R is a heteroatom-unsubstitutedC_(n)-alkenyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkenylthio” refers to a group, having thestructure —SR, in which R is a heteroatom-substituted C_(n)-alkenyl, asthat term is defined above.

The term “alkynylthio” includes straight-chain alkynylthio,branched-chain alkynylthio, cycloalkynylthio, cyclic alkynylthio,heteroatom-unsubstituted alkynylthio, heteroatom-substitutedalkynylthio, heteroatom-unsubstituted C_(n)-alkynylthio, andheteroatom-substituted C_(n)-alkynylthio. The term“heteroatom-unsubstituted C_(n)-alkynylthio” refers to a group, havingthe structure —SR, in which R is a heteroatom-unsubstitutedC_(n)-alkynyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkynylthio” refers to a group, having thestructure —SR, in which R is a heteroatom-substituted C_(n)-alkynyl, asthat term is defined above.

The term “arylthio” includes heteroatom-unsubstituted arylthio,heteroatom-substituted arylthio, heteroatom-unsubstitutedC_(n)-arylthio, heteroatom-substituted C_(n)-arylthio, heteroarylthio,and heterocyclic arylthio groups. The term “heteroatom-unsubstitutedC_(n)-arylthio” refers to a group, having the structure —SAr, in whichAr is a heteroatom-unsubstituted C_(n)-aryl, as that term is definedabove. The group, —SC₆H₅, is an example of a heteroatom-unsubstitutedarylthio group. The term “heteroatom-substituted C_(n)-arylthio” refersto a group, having the structure —SAr, in which Ar is aheteroatom-substituted C_(n)-aryl, as that term is defined above.

The term “aralkylthio” includes heteroatom-unsubstituted aralkylthio,heteroatom-substituted aralkylthio, heteroatom-unsubstitutedC_(n)-aralkylthio, heteroatom-substituted C_(n)-aralkylthio,heteroaralkylthio, and heterocyclic aralkylthio groups. The term“heteroatom-unsubstituted C_(n)-aralkylthio” refers to a group, havingthe structure —SAr, in which Ar is a heteroatom-unsubstitutedC_(n)-aralkyl, as that term is defined above. The group, —SCH₂C₆H₅, isan example of a heteroatom-unsubstituted aralkyl group. The term“heteroatom-substituted C_(n)-aralkylthio” refers to a group, having thestructure —SAr, in which Ar is a heteroatom-substituted C_(n)-aralkyl,as that term is defined above.

The term “acylthio” includes straight-chain acylthio, branched-chainacylthio, cycloacylthio, cyclic acylthio, heteroatom-unsubstitutedacylthio, heteroatom-substituted acylthio, heteroatom-unsubstitutedC_(n)-acylthio, heteroatom-substituted C_(n)-acylthio, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylategroups. The term “heteroatom-unsubstituted C_(n)-acylthio” refers to agroup, having the structure —SAc, in which Ac is aheteroatom-unsubstituted C_(n)-acyl, as that term is defined above. Thegroup, —SCOCH₃, is an example of a heteroatom-unsubstituted acylthiogroup. The term “heteroatom-substituted C_(n)-acylthio” refers to agroup, having the structure —SAc, in which Ac is aheteroatom-substituted C_(n)-acyl, as that term is defined above.

The term “alkylsilyl” includes straight-chain alkylsilyl, branched-chainalkylsilyl, cycloalkylsilyl, cyclic alkylsilyl, heteroatom-unsubstitutedalkylsilyl, heteroatom-substituted alkylsilyl, heteroatom-unsubstitutedC_(n)-alkylsilyl, and heteroatom-substituted C_(n)-alkylsilyl. The term“heteroatom-unsubstituted C_(n)-alkylsilyl” refers to a radical, havinga single silicon atom as the point of attachment, further having one,two, or three saturated carbon atoms attached to the silicon atom,further having a linear or branched, cyclic or acyclic structure,containing a total of n carbon atoms, all of which are nonaromatic, 5 ormore hydrogen atoms, a total of 1 silicon atom, and no additionalheteroatoms. For example, a heteroatom-unsubstituted C₁-C₁₀-alkylsilylhas 1 to 10 carbon atoms. An alkylsilyl group includes dialkylaminogroups. The groups, —Si(CH₃)₃ and —Si(CH₃)₂C(CH₃)₃, are non-limitingexamples of heteroatom-unsubstituted alkylsilyl groups. The term“heteroatom-substituted C_(n)-alkylsilyl” refers to a radical, having asingle silicon atom as the point of attachment, further having at leastone, two, or three saturated carbon atoms attached to the silicon atom,no carbon-carbon double or triple bonds, further having a linear orbranched, cyclic or acyclic structure, further having a total of ncarbon atoms, all of which are nonaromatic, 0, 1, or more than onehydrogen atom, and at least one additional heteroatom, that is, inaddition to the silicon atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₁-C₁₀-alkylsilyl has 1 to 10 carbon atoms.

The term “phosphonate” includes straight-chain phosphonate,branched-chain phosphonate, cyclophosphonate, cyclic phosphonate,heteroatom-unsubstituted phosphonate, heteroatom-substitutedphosphonate, heteroatom-unsubstituted C_(n)-phosphonate, andheteroatom-substituted C_(n)-phosphonate. The term“heteroatom-unsubstituted C_(n)-phosphonate” refers to a radical, havinga single phosphorous atom as the point of attachment, further having alinear or branched, cyclic or acyclic structure, further having a totalof n carbon atoms, 2 or more hydrogen atoms, a total of three oxygenatom, and no additional heteroatoms. The three oxygen atoms are directlyattached to the phosphorous atom, with one of these oxygen atoms doublybonded to the phosphorous atom. For example, a heteroatom-unsubstitutedC₀-C₁₀-phosphonate has 0 to 10 carbon atoms. The groups, —P(O)(OH)₂,—P(O)(OH)OCH₃, —P(O)(OH)OCH₂CH₃, —P(O)(OCH₃)₂, and —P(O)(OH)(OC₆H₅) arenon-limiting examples of heteroatom-unsubstituted phosphonate groups.The term “heteroatom-substituted C_(n)-phosphonate” refers to a radical,having a single phosphorous atom as the point of attachment, furtherhaving a linear or branched, cyclic or acyclic structure, further havinga total of n carbon atoms, 2 or more hydrogen atoms, three or moreoxygen atoms, three of which are directly attached to the phosphorousatom, with one of these three oxygen atoms doubly bonded to thephosphorous atom, and further having at least one additional heteroatomin addition to the three oxygen atoms, wherein each additionalheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstitutedC₀-C₁₀-phosphonate has 0 to 10 carbon atoms.

The term “phosphinate” includes straight-chain phosphinate,branched-chain phosphinate, cyclophosphinate, cyclic phosphinate,heteroatom-unsubstituted phosphinate, heteroatom-substitutedphosphinate, heteroatom-unsubstituted C_(n)-phosphinate, andheteroatom-substituted C_(n)-phosphinate. The term“heteroatom-unsubstituted C_(n)-phosphinate” refers to a radical, havinga single phosphorous atom as the point of attachment, further having alinear or branched, cyclic or acyclic structure, further having a totalof n carbon atoms, 2 or more hydrogen atoms, a total of two oxygen atom,and no additional heteroatoms. The two oxygen atoms are directlyattached to the phosphorous atom, with one of these oxygen atoms doublybonded to the phosphorous atom. For example, a heteroatom-unsubstitutedC₀-C₁₀-phosphinate has 0 to 10 carbon atoms. The groups, —P(O)(OH)H,—P(O)(OH)CH₃, —P(O)(OH)CH₂CH₃, —P(O)(OCH₃)CH₃, and —P(O)(OC₆H₅)H arenon-limiting examples of heteroatom-unsubstituted phosphinate groups.The term “heteroatom-substituted C_(n)-phosphinate” refers to a radical,having a single phosphorous atom as the point of attachment, furtherhaving a linear or branched, cyclic or acyclic structure, further havinga total of n carbon atoms, 2 or more hydrogen atoms, two or more oxygenatoms, two of which are directly attached to the phosphorous atom, withone of these two oxygen atoms doubly bonded to the phosphorous atom, andfurther having at least one additional heteroatom in addition to the twooxygen atoms, wherein each additional heteroatom is independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.For example, a heteroatom-unsubstituted C₀-C₁₀-phosphinate has 0 to 10carbon atoms.

Any apparently unfulfilled valency is to be understood to be properlyfilled by hydrogen atom(s). For example, a compound with a substituentof —O or —N is to be understood to be —OH or —NH₂, respectively.

Any genus, subgenus, or specific compound discussed herein isspecifically contemplated as being excluded from any embodimentdescribed herein.

Compounds described herein may be prepared synthetically usingconventional organic chemistry methods known to those of skill in theart and/or are commercially available (e.g., ChemBridge Co., San Diego,Calif.).

The claimed invention is also intended to encompass salts of any of thecompounds of the present invention. The term “salt(s)” as used herein,is understood as being acidic and/or basic salts formed with inorganicand/or organic acids and bases. Zwitterions (internal or inner salts)are understood as being included within the term “salt(s)” as usedherein, as are quaternary ammonium salts such as alkylammonium salts.Nontoxic, pharmaceutically acceptable salts are preferred, althoughother salts may be useful, as for example in isolation or purificationsteps during synthesis. Salts include, but are not limited to, sodium,lithium, potassium, amines, tartrates, citrates, hydrohalides,phosphates and the like. A salt may be a pharmaceutically acceptablesalt, for example. Thus, pharmaceutically acceptable salts of compoundsof the present invention are contemplated.

The term “pharmaceutically acceptable salts,” as used herein, refers tosalts of compounds of this invention that are substantially non-toxic toliving organisms. Typical pharmaceutically acceptable salts includethose salts prepared by reaction of a compound of this invention with aninorganic or organic acid, or an organic base, depending on thesubstituents present on the compounds of the invention.

Non-limiting examples of inorganic acids which may be used to preparepharmaceutically acceptable salts include: hydrochloric acid, phosphoricacid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acidand the like. Examples of organic acids which may be used to preparepharmaceutically acceptable salts include: aliphatic mono- anddicarboxylic acids, such as oxalic acid, carbonic acid, citric acid,succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphaticand aromatic sulfuric acids and the like. Pharmaceutically acceptablesalts prepared from inorganic or organic acids thus includehydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate,sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide,hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate,p-toluenesulfonate, methanesulfonate, maleate, and the like.

Suitable pharmaceutically acceptable salts may also be formed byreacting the agents of the invention with an organic base such asmethylamine, ethylamine, ethanolamine, lysine, ornithine and the like.

Pharmaceutically acceptable salts include the salts formed betweencarboxylate or sulfonate groups found on some of the compounds of thisinvention and inorganic cations, such as sodium, potassium, ammonium, orcalcium, or such organic cations as isopropylammonium,trimethylammonium, tetramethylammonium, and imidazolium.

Derivatives of compounds of the present invention are also contemplated.In certain aspects, “derivative” refers to a chemically modifiedcompound that still retains the desired effects of the compound prior tothe chemical modification. Such derivatives may have the addition,removal, or substitution of one or more chemical moieties on the parentmolecule. Non-limiting examples of the types modifications that can bemade to the compounds and structures disclosed herein include theaddition or removal of lower alkanes such as methyl, ethyl, propyl, orsubstituted lower alkanes such as hydroxymethyl or aminomethyl groups;carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, andazo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl,sulfoxido, phosphate, phosphono, phosphoryl groups, and halidesubstituents. Additional modifications can include an addition or adeletion of one or more atoms of the atomic framework, for example,substitution of an ethyl by a propyl; substitution of a phenyl by alarger or smaller aromatic group. Alternatively, in a cyclic or bicyclicstructure, heteroatoms such as N, S, or O can be substituted into thestructure instead of a carbon atom.

Compounds employed in methods of the invention may contain one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. Compounds may occur as racemates andracemic mixtures, single enantiomers, diastereomeric mixtures andindividual diastereomers. In some embodiments, a single diastereomer isobtained. The chiral centers of the compounds of the present inventioncan have the S- or the R-configuration, as defined by the IUPAC 1974Recommendations. Compounds may be of the D- or L-form, for example. Itis well known in the art how to prepare and isolate such opticallyactive forms. For example, mixtures of stereoisomers may be separated bystandard techniques including, but not limited to, resolution of racemicform, normal, reverse-phase, and chiral chromatography, preferentialsalt formation, recrystallization, and the like, or by chiral synthesiseither from chiral starting materials or by deliberate synthesis oftarget chiral centers.

In addition, atoms making up the compounds of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C.

As noted above, compounds of the present invention may exist in prodrugform. As used herein, “prodrug” is intended to include any covalentlybonded carriers which release the active parent drug or compounds thatare metabolized in vivo to an active drug or other compounds employed inthe methods of the invention in vivo when such prodrug is administeredto a subject. Since prodrugs are known to enhance numerous desirablequalities of pharmaceuticals (e.g., solubility, bioavailability,manufacturing, etc.), the compounds employed in some methods of theinvention may, if desired, be delivered in prodrug form. Thus, theinvention contemplates prodrugs of compounds of the present invention aswell as methods of delivering prodrugs. Prodrugs of the compoundsemployed in the invention may be prepared by modifying functional groupspresent in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to the parentcompound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a subject, cleaves to form a freehydroxyl, free amino, or carboxylic acid, respectively. Other examplesinclude, but are not limited to, acetate, formate, and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable. Additional examples ofpharmaceutically acceptable salts and their methods of preparation anduse are presented in Handbook of Pharmaceutical Salts: Properties,Selection and Use (2002), which is incorporated herein by reference.

E. Nucleic Acids

The present invention concerns polynucleotides and nucleic acids thatcan be used to alter the nucleic acid sequence of endogenous DNA throughhomologous recombination. These polynucleotides or nucleic acidmolecules may be isolated and/or purified from cells or a cell-extractin embodiments of the invention.

Exogenous nucleic acids that may be used in the invention include, butare not limited to, nucleic acids of the following lengths or at leastthe following lengths: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050,1060, 1070, 1080, 1090, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200,4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400,5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600,6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800,7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000,9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 10100,10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100,11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000 or morenucleotides, nucleosides, or base pairs.

“Isolated substantially away from other coding sequences” means that thegene of interest forms part of the coding region of the nucleic acidsegment, and that the segment does not contain large portions ofnaturally-occurring coding nucleic acid, such as large chromosomalfragments or other functional genes or cDNA coding regions. Of course,this refers to the nucleic acid segment as originally isolated, and doesnot exclude genes or coding regions later added to the segment by humanmanipulation.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that containsequences that will be incorporated into a host cell's DNA throughhomologous recombination.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating DNA sequences that are tobe reflected in the host cell's genome through homologous recombination.

Vectors of the present invention are designed, primarily, to transformcells with a desired sequence that can lead to the introduction ordeletion of all or part of that sequence. In some embodiments, this mayinvolve a sequence under the control of a eukaryotic promoter (i.e.,constitutive, inducible, repressable, tissue specific). Also, thevectors may contain a selectable marker if, for no other reason, tofacilitate their manipulation in vitro. However, selectable markers mayplay an important role in producing recombinant cells.

Tables 1 and 2, below, list a variety of regulatory signals for useaccording to the present invention.

TABLE 1 Inducible Elements Element Inducer References MT II PhorbolEster (TPA) Palmiter et al., 1982; Haslinger and Heavy metals Karin,1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987,Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse Glucocorticoids Huang et al., 1981; Lee et al., 1981; mammarytumor Majors and Varmus, 1983; Lee et virus) al., 1984; Ponta et al.,1985 β-Interferon poly(rI)x Tavernier et al., 1983 poly(rc) Adenovirus 5E2 Ela Imperiale and Nevins, 1984 Collagenase Phorbol Ester (TPA) Angelet al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40Phorbol Ester (TFA) Angel et al., 1987b Murine MX Gene Interferon,Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187 Resendez etal., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin SerumRittling et al., 1989 MHC Class I Gene Interferon Blanar et al., 1989H-2κb HSP70 Ela, SV40 Large T Taylor et al., 1989; Taylor and AntigenKingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989Tumor Necrosis Factor MA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

TABLE 2 Other Promoter/Enhancer Elements Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl and Baltimore, 1985; Atchison and Perry, 1986, 1987; Imler etal., 1987; Neuberger et al., 1988; Kiledjian et al., 1988;Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard andSchaffner, 1985 T-Cell Receptor Luria et al., 1987, Winoto andBaltimore, 1989; Redondo et al., 1990 HLA DQ α and DQ β Sullivan andPeterlin, 1987 β-Interferon Goodbourn et al., 1986; Fujita et al., 1987;Goodbourn and Maniatis, 1985 Interleukin-2 Greene et al., 1989Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC ClassII 5 Koch et al., 1989 MHC Class II HLA-Drα Sherman et al., 1989 β-ActinKawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase Jaynes etal., 1988; Horlick and Benfield, 1989; Johnson et al., 1989a Prealbumin(Transthyretin) Costa et al., 1988 Elastase I Ornitz et al., 1987Metallothionein Karin et al., 1987; Culotta and Hamer, 1989 CollagenasePinkert et al., 1987; Angel et al., 1987 Albumin Gene Pinkert et al.,1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al., 1988;Campere and Tilghman, 1989 γ-Globin Bodine and Ley, 1987; Perez-Stableand Constantini, 1990 β-Globin Trudel and Constantini, 1987 c-fos Cohenet al., 1987 c-HA-ras Treisman, 1985; Deschamps et al., 1985 InsulinEdlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990(NCAM) a₁₋Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang etal., 1990 Mouse or Type I Collagen Rippe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji etal., 1981; Moreau et al., 1981; Sleigh and Lockett, 1985; Firak andSubramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadeschand Berg, 1986; Wang and Calame, 1986; Ondek et al., 1987; Kuhl et al.,1987; Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975;Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndall et al., 1981;Dandolo et al., 1983; Hen et al., 1986; Campbell and Villarreal, 1988Retroviruses Kriegler and Botchan, 1983; Kriegler et al., 1984a, b,;Bosze et al., 1986; Miksicek et al., 1986; Celander and Haseltine, 1987;Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1996; Reismanand Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan,1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987;Stephens and Hentschel, 1987 Hepatitis B Virus Bulla and Siddiqui, 1988;Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee,1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber andCullen, 1988; Jakobovits et al., 1988; Feng and Holland, 1988; Takebe etal., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp andMarciniak, 1989; Braddock et al., 1989 Cytomegalovirus Weber et al.,1984; Boshart et al., 1985; Foecking and Hofstetter, 1986 Gibbon ApeLeukemia Virus Holbrook et al., 1987; Quinn et al., 1989

The promoters and enhancers that control the transcription of proteinencoding genes in eukaryotic cells are composed of multiple geneticelements. The cellular machinery is able to gather and integrate theregulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

The term “promoter” will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between elements is flexible, so that promoterfunction is preserved when elements are inverted or moved relative toone another. In the tk promoter, the spacing between elements can beincreased to 50 bp apart before activity begins to decline. Depending onthe promoter, it appears that individual elements can function eitherco-operatively or independently to activate transcription.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Aside from this operational distinction, enhancers and promoters arevery similar entities.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

In some embodiments, the promoter for use in the present invention isthe cytomegalovirus (CMV) immediate early (IE) promoter. This promoteris commercially available from Invitrogen in the vector pcDNAIII, whichis some for use in the present invention. Also contemplated as useful inthe present invention are the dectin-1 and dectin-2 promoters. Below area list of additional viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thepresent invention. Additionally any promoter/enhancer combination (asper the Eukaryotic Promoter Data Base EPDB) could also be used to driveexpression of structural genes encoding oligosaccharide processingenzymes, protein folding accessory proteins, selectable marker proteinsor a heterologous protein of interest.

Another signal that may prove useful is a polyadenylation signal. Suchsignals may be obtained from the human growth hormone (hGH) gene, thebovine growth hormone (BGH) gene, or SV40.

The use of internal ribosome binding sites (IRES) elements are used tocreate multigene, or polycistronic, messages. IRES elements are able tobypass the ribosome scanning model of 5-methylatd cap-dependenttranslation and begin translation at internal sites (Pelletier andSonenberg, 1988). IRES elements from two members of the picornavirusfamily (polio and encephalomyocarditis) have been described (Pelletierand Sonenberg, 1988), as well an IRES from a mammalian message (Macejakand Sarnow, 1991). IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame is accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage.

In any event, it will be understood that promoters are DNA elementswhich when positioned functionally upstream of a gene leads to theexpression of that gene. Most transgene constructs of the presentinvention are functionally positioned downstream of a promoter element.

Compositions and methods of the invention are provided for administeringthe compositions of the invention to a patient.

Nucleic acids may also be comprised in a vector. The term “vector” isused to refer to a carrier nucleic acid molecule into which a nucleicacid sequence can be inserted for introduction into a cell where it canbe replicated. A nucleic acid sequence can be “exogenous,” which meansthat it is foreign to the cell into which the vector is being introducedor that the sequence is homologous to a sequence in the cell but in aposition within the host cell nucleic acid in which the sequence isordinarily not found. Vectors include plasmids, cosmids, viruses(bacteriophage, animal viruses, and plant viruses), and artificialchromosomes (e.g., YACs). One of skill in the art would be well equippedto construct a vector through standard recombinant techniques, which aredescribed in Sambrook et al., (2001) and Ausubel et al., 1996, bothincorporated herein by reference. In addition to encoding a modifiedpolypeptide such as modified gelonin, a vector may encode non-modifiedpolypeptide sequences such as a tag or targetting molecule. Usefulvectors encoding such fusion proteins include pIN vectors (Inouye etal., 1985), vectors encoding a stretch of histidines, and pGEX vectors,for use in generating glutathione S-transferase (GST) soluble fusionproteins for later purification and separation or cleavage. A targettingmolecule is one that directs the modified polypeptide to a particularorgan, tissue, cell, or other location in a subject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described infra.

Protamine may also be used to form a complex with an expressionconstruct. Such complexes may then be formulated with the lipidcompositions described above for administration to a cell. Protaminesare small highly basic nucleoproteins associated with DNA. Their use inthe delivery of nucleic acids is described in U.S. Pat. No. 5,187,260,which is incorporated by reference.

The following are methods of recombinant gene delivery to a given hostcell and are thus considered consistent with the present invention.

1. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

2. Knockout Animals

The generation of an animal model lacking a particular genetic sequenceis considered part of the invention. The lack of genetic sequences mayprovoke various types of pathophysiological disturbances in a knockoutanimal. One method of inhibiting the endogenous expression of a gene inan animal is to disrupt the gene in germline cells and produce offspringfrom these cells. This method is generally known as knockout technology.U.S. Pat. No. 5,616,491, incorporated herein by reference in itsentirety, generally describes the techniques involved in the preparationof knockout mice, and in particular describes mice having a suppressedlevel of expression of the gene encoding CD28 on T cells, and micewherein the expression of the gene encoding CD45 is suppressed on Bcells. Pfeffer et al. (1993) describe mice in which the gene encodingthe tumor necrosis factor receptor p55 has been suppressed. The miceshowed a decreased response to tumor necrosis factor signaling.Fung-Leung et al. (1991a; 1991b) describe knockout mice lackingexpression of the gene encoding CD8. These mice were found to have adecreased level of cytotoxic T cell response to various antigens and tocertain viral pathogens such as lymphocytic choriomeningitis virus.

The term “knockout” refers to a partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence in a cell. The term “knockout construct” refers to anucleic acid sequence that is designed to decrease or suppressexpression of a protein encoded by endogenous DNA sequences in a cell.The nucleic acid sequence used as the knockout construct is typicallycomprised of: (1) DNA from some portion of the gene (exon sequence,intron sequence, and/or promoter sequence) to be suppressed; and (2) amarker sequence used to detect the presence of the knockout construct inthe cell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native DNA sequence. Such insertionusually occurs by homologous recombination (i.e., regions of theknockout construct that are homologous to endogenous DNA sequenceshybridize to each other when the knockout construct is inserted into thecell and recombine so that the knockout construct is incorporated intothe corresponding position of the endogenous DNA).

The knockout construct nucleic acid sequence may comprise 1) a full orpartial sequence of one or more exons and/or introns of the gene to besuppressed, 2) a full or partial promoter sequence of the gene to besuppressed, or 3) combinations thereof. Typically, the knockoutconstruct is inserted into an embryonic stem cell (ES cell) and isintegrated into the ES cell genomic DNA, usually by the process ofhomologous recombination. This ES cell is then injected into, andintegrates with, the developing embryo.

Furthermore, knock-out mice having any phenotype that resembles adisease state may be used to screen or test therapeutic drugs that slow,modify, or cure conditions. As is known to the skilled artisan, aconditional knockout, wherein the gene is disrupted under certainconditions, is frequently used.

3. Transgenic Animals

It is further contemplated that transgenic animals are part of thepresent invention. A transgenic animal of the present invention mayinvolve an animal created using methods of the invention.

In a general aspect, a transgenic animal is produced by the integrationof a given transgene into the genome in a manner that permits theexpression of the transgene, or by disrupting the wild-type gene,leading to a knockout of the wild-type gene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. (1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

U.S. Pat. No. 5,639,457 is also incorporated herein by reference tosupplement the present teaching regarding transgenic pig and rabbitproduction. U.S. Pat. Nos. 5,175,384; 5,175,385; 5,530,179, 5,625,125,5,612,486 and 5,565,186 are also each incorporated herein by referenceto similarly supplement the present teaching regarding transgenic mouseand rat production. Transgenic animals may be crossed with othertransgenic animals or knockout animals to evaluate phenotype based oncompound alterations in the genome.

F. Pharmaceutical Formulations and Administration Thereof

1. Pharmaceutical Formulations and Routes of Administration

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more candidate substance or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one candidatesubstance or additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the therapeutic or pharmaceutical compositions is contemplated.

The compounds of the invention may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, systemically, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, locally, via inhalation (e.g., aerosolinhalation), via injection, via infusion, via continuous infusion, vialocalized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the foregoing as would be known to one ofordinary skill in the art (see, for example, Remington's PharmaceuticalSciences, 1990).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of a compound of the present invention. Inother embodiments, the compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal, or combinations thereof.

The candidate substance may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine, or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Itmay be preferable to include isotonic agents, such as, for example,sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in certain embodiments the aqueous nasal solutions usually are isotonicor slightly buffered to maintain a pH of about 5.5 to about 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the candidate substance is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet. Incertain embodiments, carriers for oral administration comprise inertdiluents, assimilable edible carriers or combinations thereof. In otheraspects of the invention, the oral composition may be prepared as asyrup or elixir. A syrup or elixir, and may comprise, for example, atleast one active agent, a sweetening agent, a preservative, a flavoringagent, a dye, a preservative, or combinations thereof.

In certain embodiments an oral composition may comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina, or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides, or combinations thereof In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, certain methodsof preparation may include vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin, or combinations thereof.

2. Combination Therapy

In some embodiments, it is contemplated that the RAD51 modulators of theinvention may be used in conjunction with DNA damaging agents as part ofa treatment regimen. This process may involve contacting the cell(s)with the agents at the same time or within a period of time whereinseparate administration of the agents produces a desired therapeuticbenefit. This may be achieved by contacting the cell, tissue or organismwith a single composition or pharmacological formulation that includestwo or more agents, or by contacting the cell with two or more distinctcompositions or formulations, wherein one composition includes one agentand the other includes another.

The compounds of the present invention may precede, be co-current withand/or follow the other agents by intervals ranging from minutes toweeks. In embodiments where the agents are applied separately to a cell,tissue or organism, one would generally ensure that a significant periodof time did not expire between the time of each delivery, such that theagents would still be able to exert an advantageously combined effect onthe cell, tissue or organism. For example, in such instances, it iscontemplated that one may contact the cell, tissue or organism with two,three, four or more modalities substantially simultaneously (i.e.,within less than about a minute) as the candidate substance. In otheraspects, one or more DNA damaging agents may be administered or providedwithin 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any rangederivable therein, prior to and/or after administering the RAD51modulator.

Various combination regimens of the agents may be employed. Non-limitingexamples of such combinations are shown below, wherein a compound of thepresent invention is “A” and a second agent, such as a DNA damagingagent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

In some embodiments, more than one course of therapy may be employed. Itis contemplated that multiple courses may be implemented. In certainembodiments, a patient may have previously undergone radiation orchemotherapy for a cancer that turns out to be chemotherapy- orradiation-resistant. Alternatively, a patient may have a recurringcancer that is to be treated with a DNA damaging agent.

G. Organisms and Cell Source

Cells that may be used in many methods of the invention can be from avariety of sources. Embodiments include the use of mammalian cells, suchas cells from monkeys, chimpanzees, rabbits, mice, rats, ferrets, dogs,pigs, humans, and cows. Alternatively, the cells may be from fruitflies, yeast, or e. coli, which are all model systems for evaluatinghomologous recombination.

Methods of the invention can involve cells, tissues, or organs involvingthe heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein,artery, cornea, blood, small intestine, large intestine, brain, spinalcord, smooth muscle, skeletal muscle, ovary, testis, uterus, andumbilical cord.

Moreover, methods can be employed in cells of the following type:platelet, myelocyte, erythrocyte, lymphocyte, adipocyte, fibroblast,epithelial cell, endothelial cell, smooth muscle cell, skeletal musclecell, endocrine cell, glial cell, neuron, secretory cell, barrierfunction cell, contractile cell, absorptive cell, mucosal cell, limbuscell (from cornea), stem cell (totipotent, pluripotent or multipotent),unfertilized or fertilized oocyte, or sperm.

Moreover, methods can be implemented with or in plants or parts ofplants, including fruit, flowers, leaves, stems, seeds, cuttings. Plantscan be agricultural, medicinal, or decorative.

H. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Etheno DNA-Based Assay for RAD51 Filament Formation

RAD51 filament formation is a well accepted critical step in theinitiation of HR repair. Biochemical assays have been developed tomeasure RAD51 protein assembly on sites of single stranded DNA (ssDNA)in vitro. Most studies have used etheno DNA (also called epsilon DNA) asthe substrate in this assay. Etheno DNA is formed in a chemical reactionthat has been recognized since the early 1970's (Secrist et al., 1972;Barrio et al., 1972). In this reaction, single stranded DNA (commonlyphage DNA or an oligonucleotide) is incubated with choroacetaldehyde ina low pH solution. The chemical intermediates in this reaction arematured by further incubation at elevated temperatures (Krzyzosiak etal., 1981). This generates a chemical modification of the cytidine andadenosine bases, resulting in fluorescent ethenocytidine andethenoadenosine derivatives.

Alternatively, oligonucleotides can be directly synthesized in theetheno form (typically poly-etheno dA). Numerous studies over the past25 years have reported that the fluorescent signal emitted by etheno DNAis increased when bound by proteins. The level to which proteins bindetheno DNA can be quantitatively measured by fluorescencespectrophotometry. When saturated with an excess of a DNA-bindingprotein, the fluorescence above baseline increases approximately 2-4fold. This technique has been used widely to characterize the conditionsand kinetics for a variety of DNA-interacting proteins and compounds(Zaitseva et al., 1999; Menetski and Kowalczykowski, 1985; Sugiyama etal., 1997). The inventors have used this technique to confirm theactivity of purified RAD51 protein and lack thereof in the RAD51 (F86E)mutant.

Example 2 Fluorescence Polarization Assay for RAD51 Filament Formation

RAD51 filament formation can also be studied using oligonucleotides thatare end-labeled with a fluorescent tag. Binding of proteins to thissubstrate DNA can be detected as an increase in anisotropy (orpolarization) of the fluorescent tag. This method has been used tomonitor the DNA-binding activity of yeast RAD51 (Kim et al., 2001) andRecA (Wittung et al., 1997; Ellouze et al., 1999), which is theeukaryotic homolog of RAD51. In this example, this method was modifiedso that very small sample volumes (30 μl) can be monitored in ahigh-throughput scale, using 384-well plates and a fluorescent platereader. A comparison of the two methods shows essentially identicalresults in term of RAD51 binding. However, the fluorescence polarizationassay is less expensive, faster, and it generates results with superiorsignal to background ratios (FIGS. 2A-2B). By varying the amount ofsubstrate DNA, a threshold of approximately 0.1 μM RAD51 protein isrequired for in vitro filament formation in this system. Furthermore,RAD51 filament formation in this assay is more efficient and/or morestable in buffers containing calcium ions. This is consistent with aprior report showing that calcium stimulates yeast RAD51 in thisfluorescence polarization assay, and renders filaments more stable inhigh salt buffers (Kim et al., 2001). Another report showed that calciuminhibits ATP hydrolysis by human RAD51, resulting in stabilization offilaments and stimulation of strand exchange activity (Bugreev andMazin, 2004).

Example 3 Peptide-Alkaline Phosphatase (AP) Fusion-Based Assay forDetection of RAD51-Protein Interactions

An assay to quantitatively measure protein-protein interactions using apeptide-alkaline phosphatase (AP) fusion system was developed (Han etal., 2004). In this system, oligonucleotide duplexes encoding peptidesequences are cloned into an AP expression plasmid (pEZ707, gift of Dr.Brian Kay). The plasmids are transformed into BL21(DE3) bacteria,expressed, and purified. Streptavidin-coated plates (96-well format) arecoated with a biotinylated bait protein. The binding of peptide-APfusion proteins to bait-coated wells are quantified with a plate readerfollowing incubation with an alkaline phosphatase substrate (FIG. 3,left). This system allows for a large number of peptide sequences to berapidly screened.

Example 4 Assay for Measuring Interactions Between RAD51 and BRC4

A fusion protein was prepared, such that full-length BRC4 (BRCA2residues 1511-1579) was linked to the N-terminus of alkalinephosphatase. This 69 amino acid wild-type polypeptide exhibited bindingto RAD51-coated wells and minimal interaction with uncoated wells (FIG.3, right). The binding was about 4-fold stronger to RAD51(F86E) coatedwells, relative to wild-type RAD51 coated wells. This F86E mutation isknown to impair RAD51's ability to self-associate (Pellegrini et al.,2002; Yu et al., 2003); consequently the RAD51(F86E) protein probablyserves as a better ‘bait’ in this assay because it is composed primarilyof protein monomers. A mutant BRC4-AP fusion was also preparedcontaining an alanine substitution at a residue corresponding totyrosine-1526 of BRCA2. This BRC4(T1526A) mutant showed markedly reducedbinding, relative to wild type BRC4. This result is consistent withprevious studies that examined analogous T to A mutations in BRC repeatsusing other types of assays (Chen et al., 1999; Davies et al., 2001).

Example 5 Determination of the Minimal Length of BRC4 Sufficient forBinding to RAD51

As discussed previously, the conditional expression of BRC4 polypeptidein a breast cancer cell line results in a hypersensitivity to DNA damage(Chen et al., 1999). One possible consideration is for inhibiting HRwith a BRC4-based peptide that can be synthetically prepared anddirectly delivered from tissue culture media into cells. An expectedproblem with this plan relates to the relatively large size of BRC4 (69amino acids), which could present challenges in terms of large-scalesynthetic peptide preparation. However, studies focusing on other BRCrepeats have shown that the entire polypeptide may not always berequired for RAD51 binding (Chen et al., 1999; Chen et al., 1998; Wonget al., 1997).

For this reason, the AP-fusions system was used to determine the minimallength of BRC4 sufficient for binding to RAD51. Several AP-fusionproteins were prepared such that residues were truncated from eitherside of BRC4 in a step-wise manner (FIG. 4). These were tested for theability to bind RAD51(F83E) coated wells. These results demonstrate thatC-terminal truncations beyond the aspartic acid residue (correspondingto amino acid 1547 of BRCA2) markedly reduce binding; while smallerC-terminal truncations had no significant impact on binding. Similarly,up to 12 residues could be truncated from the N-terminus of BRC4 withoutimpacting binding. N-terminal truncations were not attempted beyond theglycine (corresponding to amino acid 1523 of BRCA2), since this regioncontains the conserved oligomerization motif. Thus the minimal lengthsufficient for binding was determined to be amino acids 1523-1547 ofBRCA2.

An alanine substitution at a residue corresponding to tyrosine-1526 ofBRCA2 resulted in markedly reduced binding of this minimal peptide,consistent with the mutagenesis results in full length BRC4. An F1524Esubstitution in BRC4 resulted in even greater loss of RAD51 binding. Apeptide fragment of RAD51 (residues 85-110) corresponding to the minimalBRC4 peptide was prepared in the AP fusion system. This peptide hadconsiderably less binding to full length RAD51 (F86E), relative to theminimal BRC4 peptide. Similar to the BRC4 result, an analogous mutation(F86E) in the RAD51 fragment reduced binding to full-length RAD51(F86E).

Example 6 Identification of RAD51 Enhancers

The assay of Example 2 was used to serve in the screen of asmall-molecule chemical library for compounds that stimulate RAD51filament formation. For the screen conditions that resulted in 50%saturation of the FP endpoint were selected. Multiple positive (+RAD51)and negative (RAD51) controls were tested, and these readings weretested using the statistical method described by Zhang et al. (1999),which evaluates the suitability of an HT assay. The assay as describedhas a Z-factor of 0.82, validating it as an excellent assay for HTscreening.

A naïve 10,000 compound library of small molecules (Chembridge DIVERSetcollection) was screened. A ‘mixed compound’ screening strategy wasused, wherein each reaction tested a mixture of 8 compounds. Whenfilament stimulation was achieved for any given compound mixture, the 8compounds from that mixture were individually tested using the samecompound concentration. This identified 3 small molecule compounds thatstimulate RAD51 filament formation by at least 50% (FIGS. 8A-8E,compounds 45488 (also called compound 5253121), 43783 and 41936).Further study of the most the active compound (45488) (also calledcompound 5253121) confirmed that it enhances RAD51 filament formation,and that it protects these filaments from buffers containing high saltconcentrations (which typically destabilize RAD51 filaments). Imagingwith electron microscopy has confirmed that the increases in measured FPare, in fact, due to compound-stimulated filaments with long tracklengths (FIG. 5). These experiments have been repeated with RecA protein(the bacterial ortholog of RAD51) and demonstrated no stimulation offilament formation, suggesting the compound effect is specific to RAD51.

This compound was also tested using an assay that tests strand invasion,a later step in HR (FIG. 6A). Briefly, 15 μl reaction volumes included aDNA strand exchange protein (0.8 μM) that was pre-incubated for 5 min at37° C. with 1 μM (nucleotide concentration) ³²P-labeled oligonucleotidein a reaction buffer containing 20 mM Hepes (pH 7.5), 1 mM DTT, 2 mMnucleotide cofactor, and 1 mM MgCl₂, and various concentrations of RS-1.For experimental buffer conditions that included calcium, 1 mM CaCl₂ waspresent in addition to (in the case of hRAD51) or in the place of (inthe case of RecA and scRAD51) the 1 mM MgCl₂. Conditions with scRAD51additionally contained 110 nM scRAD54. Following this initial bindingreaction, 10 μl of 19.75 μM (base pair concentration) supercoiledhomolog-containing target plasmid DNA (pRS306) was next added along withsufficient magnesium acetate to give a final concentration of 10 mM. DNAsubstrates were allowed form a homology dependent association (or‘D-loop’), and reactions were stopped with SDS and proteinase K. Theresulting DNA was separated on 1.3% agarose gels, transferred ontonitrocellulose membranes, analyzed by phosphorimaging, and quantifiedwith ImageJ 1.37v software (NIH).

These experiments demonstrated that the compound is capable ofstimulating DNA strand invasion activity of RAD51 (FIG. 6B). Theseexperiments were repeated using other recombinase proteins (bacterialRecA and yeast DMC1) in the place of human RAD51. The compound had noeffect on RecA and demonstrated some inhibition of DMC1. This furthersupports the inventor's belief that the compound's enhancery effect isRAD51 specific.

An additional 20 compounds were identified in the Cambridge library thatshared varying degrees of structural similarity to compound 45488 (alsocalled compound 5253121). These are also shown in FIGS. 8A-8E.

Example 7 Cell Survival Study Using a RAD51 Enhancer—Normal DiploidFibroblasts

Normal diploid fibroblasts were plated in 96-well format (900cells/well) and were incubated for 24 hrs with various doses ofcisplatin and 45488 compound (also called compound 5253121)(administered simultaneously). Drugs were then removed and replaced withnormal media. Cells were allowed to grow for 6 days, and were then fixedand stained with sulforhodamine B (SRB). The amount of surviving cellswas quantified as a function of OD(564) with a plate reader (FIGS.7A-7C). Each data point represents the mean of 6 repeat wells, and errorbars represent the standard error of the mean (SEM).

Example 8 Fluoresence Polarization Assay Using RAD51 Enhancers

The compounds shown in FIGS. 8A-8E were tested using the assay ofExample 2. The results are shown in FIG. 10.

Example 9 Identification of RAD51 Inhibitors

The assay of Example 2 was used to serve in the screen of asmall-molecule chemical library for compounds that inhibit RAD51filament formation. The assay performed well in black polystyrene384-well plates. FP readings were very reproducible with reactionvolumes in the 10-100 μl range, and 100 μl was typically used. The HTscreen used small molecules from the ChemBridge library collections(10,000 compound DIVERSet collection). Compounds are stored at 0.5 mg/mlconcentrations; the molecular weights vary so the average compound stockconcentration is 14.7 mM. The assay was optimized such that the positivecontrol FP reading was ˜50% of maximal reaction saturation; the RAD51protein concentration was 0.3 uM and the fluorescein-labeled DNA is at0.1 uM (nucleotide concentration). At these concentrations the positive(+RAD51) and negative (−RAD51) controls were tested in 50 independentwells each. The average FP readings for positive control and negativecontrol are 344 mP (SD=13.4) and 27 mP (SD=5.3), respectively. Thesereadings were tested using the statistical method described by Zhang etal, which evaluates the suitability of an HT assay (Zhang et al., 1999).The performance of the assay as described has a Z-factor of 0.82,suggesting that it is an excellent assay for the proposed HT screen.

The inventors used a ‘mixed compound’ screening strategy, so each wellin the test plate contained a mixture of 8 compounds. This strategy hasthe advantage of higher throughput than single-compound screening; it isbased on the low statistical probability that any one well will containboth an inhibitory and a enhancery compound, rendering their effectsundetectable. Plates of compound mixtures were prepared such thatstarting compound concentrations would be 6.25 μg/ml in each reaction(average of 18.3 μM). The compounds were manually added to RAD51 proteinin reaction buffer with a multi-channel pipettor, and the mixture waspre-incubated at room temperature for 5 minutes. The DNA substrate isthen added, plates are incubated for 30 minutes at 37°, and FP ismeasured. FP data points from each test well were normalized in twoways: 1) to the corresponding positive control for each plate; 2) to themedian compound FP value for each plate. When filament inhibition wasachieved with a compound mixture, the 8 compounds from that mixture wereindividually tested. This screen identified 3 compounds that enhancefilament formation by at least 50%. It also identified 72 compounds withat least 55% inhibition of filament formation in the standard buffercontaining 10 mM Mg²⁺ (FIG. 9A-9S). These 72 compounds were re-tested ina buffer containing 1 mM Mg²⁺ and 2 mM Ca²⁺. Reactions in this morestringent buffer condition identified 28 of the 72 that were capable ofinhibiting filament formation by 60% or more (Table 3):

TABLE 3 Plate location % Inhibition 412 10 3 65.5139 460 6 2 69.88384448 6 2 72.28592 470 5 4 73.69906 487 8 5 74.4119 497 6 8 76.83156 487 87 78.70403 534 9 8 86.57293 487 8 6 89.31386 187 11 4 89.63514 474 11 489.97149 524 5 8 92.53421 474 5 5 93.80428 469 6 7 94.15568 461 4 794.31381 509 8 6 95.112 474 11 2 95.49854 448 6 7 95.50858 520 7 296.116 520 5 1 96.27915 520 9 8 96.47493 497 3 5 97.00204 467 6 297.23045 472 8 3 97.26559 452 6 8 97.35595 452 6 4 98.13154 498 7 298.64861 476 9 3 99.02762

Example 10 Secondary Screen to Detect the Subset Capable of BlockingRAD51-RAD51 Protein Monomer Association or RAD51-BRC4 Association

The 72 inhibitory compounds discussed in Example 9 were subjected to asecondary in vitro screen to identify a sub-set of compounds whichfunction by interrupting RAD51-RAD51 protein monomer association orRAD51-BRC4 association. As discussed earlier, BRC peptides share aconserved motif that is thought to mimic the primary interface used inRAD51 monomer-monomer interactions (Shin et al., 2003; Pellegrini etal., 2002). The minimal BRC4 peptide length sufficient for thisinteraction has been identified (Example 5) and this peptide has beenprepared in fusion to alkaline phosphatase. This BRC4-AP fusion proteinbinds specifically to polystyrene wells coated with RAD51 (F86E). Notethat BRC4 peptide may be preferable over RAD51-based peptides in thisassay, because BRC4's binding is stronger than RAD51 fragments in thisassay. This was also performed using an AP fusion containing the RAD51(85-110) fragment shown in FIG. 4.

Each well of a streptavidin-coated 96-well polystyrene plate wasincubated with 2.5 μg biotinylated RAD51(F86E) protein in PBS. Unboundprotein was aspirated, and wells blocked with 5% dried milk in PBS. Eachpeptide-AP fusion protein was diluted in PBS, and 80 μl of each wasincubated in a protein-coated well for 60 minutes. Non-binding AP-fusionproteins were aspirated, and wells were washed 3 times with PBS. Thelevels of well-bound fusion protein were determined by adding 50 μlp-Nitrophenyl Phosphate (PNPP) substrate to each well and incubating atroom temperature. Kinetic values of OD405 were measured with a SynergyHT plate reader (Bio-Tek Instruments, Winooski, Vt.), and werenormalized to the specific activity of the particular AP-peptide fusion.The 72 inhibitory compounds were tested for the ability to inhibitBRC4-AP binding to RAD51(F86E), resulting in 12 compounds that inhibitbinding by at least 30% (Table 4). The 72 compounds were also tested forthe ability to inhibit RAD51(85-110)-AP binding to RAD51(F86E),resulting in 6 compounds that inhibit binding by at least 30% (Table 5).

TABLE 4 Plate location % inhibition 474 11 4 40.59041 486 11 4 70.1107497 3 5 64.20664 514 7 1 46.49446 522 10 8 74.53875 505 7 6 34.68635

TABLE 5 Plate location % inhibition 415 3 5 47.99216 426 5 7 43.095 4395 3 62.2578 460 6 2 38.62369 474 11 4 35.42989 497 3 5 69.92292 514 7 146.71464 518 3 2 48.63092 506 10 7 33.46908 451 2 3 51.53983 505 7 633.72359 535 7 1 72.91932

Example 11 Clonogenic Survival Study Using a RAD51 Enhancer in thePresence of Radiation

Single chicken lymphocyte DT40 cells were irradiated. Thirty minuteslater, they were incubated with compound 45488 (also called compound5253121) for 24 hours. Single cells were then plated (100, 1000, or10000) in 6-well plates in media. Because DT40 cells are not adherent toplastic surfaces, the media included methylcellulose to provideviscosity. After 7-10 days, the resulting colonies are counted, andsurvival is reported as a colony-forming ability relative toun-irradiated cells.

The results, seen in FIG. 12, demonstrate that compound 45488 protectsthe cells from radiation damage. The compound appears to act as amitigator of radiation damage (as opposed to a simple protector),because the compound was added to cells about 30 minutes afterirradiation of the cells.

Future experiments will entail RAD51 enhancer/radiation clonogenicsurvival assays in mutant DT40 cells that are deficient in proteins thatmediate RAD51 filament assembly at double-strand breaks. If a greatereffect of the RAD51 enhancer is observed in these cells (relative towild type cells), this will provide evidence that the RAD51 enhancerhelps cells bypass the requirement of assembly mediators.

Example 12 HR Analysis Using a GFP Recombination Report System

Assay for HR using GFP-based reporter construct: This assay wasperformed as previously described, with some modifications. Esashi etal., 2005 (incorporated herein by reference in its entirety). U2OS cellscontaining a single chomosomal copy of the DR-GFP reporter (gift ofMaria Jasin, Memorial Sloan Kettering Cancer Center) were transfectedwith an I-SceI expression vector (pCBASce), which was also a gift ofMaria Jasin. Cells were subsequently incubated in media containingcandidate compounds (20 μM unless otherwise indicated) for 24 hours,followed by an additional 24 hour incubation in normal media. Thepercentage of GFP-expressing cells were determined via flow cytometry.Dead cells were excluded by staining with 7-amino-actinomycin D. Resultsmay be seen regarding certain RAD51 inhibitors in FIGS. 15A-15C.

All of the methods and apparatuses disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand apparatuses and in the steps or in the sequence of steps of themethods described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for stimulating RAD51 protein in acell comprising providing to the cell a RAD51 stimulator that is acompound having formula (VII):

or a salt thereof, wherein R₄₅ is H, —C(O)(CH₂)_(q)NH-aryl,—NHC(O)O-alkyl, or

wherein R₄₉ is halogen or alkoxy; Z₂₀ is O or S; and q is 0-6; R₄₆ is Hor halogen; R₄₇ is H, alkyl, hydroxy, or halogen; R₄₈ is H, hydroxy, or—SO₂NH(CH₂)_(r)-aryl, wherein r is 0-6; and R₅₀ is H or hydroxy whereinat least one of R₄₅, R₄₆, R₄₇, R₄₈, or R₅₀ is not H.
 2. The method ofclaim 1, wherein the RAD51 stimulator is any one or more compounds withformula VII found in FIGS. 8A-8E, or a salt thereof.
 3. The method ofclaim 1, further comprising administering to the cell a compositioncomprising a nucleic acid under conditions that promote incorporation ofthe nucleic acid into the cell's nucleic acid.
 4. The method of claim 1,wherein the RAD51 protein stimulator is further defined as:


5. A method for incorporating exogenous DNA into cells' DNA comprising:a) transfecting the cells with a composition comprising the exogenousDNA; and, b) contacting the cells with an effective amount of a RAD51stimulator.
 6. The method of claim 5, wherein the RAD51 stimulator is acompound of formula (VII)

or a salt thereof, wherein R₄₅ is H, —C(O)(CH₂)_(q)NH-aryl,—NHC(O)O-alkyl, or

wherein R₄₉ is halogen or alkoxy; Z₂₀ is O or S; and q is 0-6; R₄₆ is Hor halogen; R₄₇ is H, alkyl, hydroxy, or halogen; R₄₈ is H, hydroxy, or—SO₂NH(CH₂)_(r)-aryl, wherein r is 0-6; and R₅₀ is H or hydroxy whereinat least one of R₄₅, R₄₆, R₄₇, R₄₈, or R₅₀ is not H.
 7. The method ofclaim 5, wherein the RAD51 stimulator is any one or more compounds foundin FIGS. 8A-8E, or a salt thereof.
 8. A method for treating DNA damagein cells comprising providing to the cell a RAD51 enhancer comprising acompound of formula (VII)

or a salt thereof, wherein R₄₅ is H, —C(O)(CH₂)_(q)NH-aryl,—NHC(O)O-alkyl, or

wherein R₄₉ is halogen or alkoxy; Z₂₀ is O or S; and q is 0-6; R₄₆ is Hor halogen; R₄₇ is H, alkyl, hydroxy, or halogen; R₄₈ is H, hydroxy, or—SO₂NH(CH₂)_(r)-aryl, wherein r is 0-6; and R₅₀ is H or hydroxy whereinat least one of R₄₅, R₄₆, R₄₇, R₄₈, or R₅₀ is not H.
 9. The method ofclaim 8, wherein the cells have been treated with or will be treatedwith a DNA damaging agent.
 10. The method of claim 9, wherein the cellsare provided with the RAD51 enhancer before being treated with a DNAdamaging agent.
 11. The method of claim 9, wherein the cells areprovided with the RAD51 enhancer after being treated with a DNA damagingagent or radiation.